JP5112863B2 - Human anti-KIR antibody - Google Patents

Abstract

Compositions and methods for regulating an immune response in a subject are described. More particularly, described are human antibodies that regulate the activity of NK cells and allow a potentiation of NK cell cytotoxicity in mammalian subjects, and antibodies having antigen-binding properties similar to those of human monoclonal antibody 1-7F9 or 14F1. Described also are fragments and derivatives of such antibodies, as well as pharmaceutical compositions comprising the same and their uses, particularly for use in therapy, to increase NK cell activity or cytotoxicity in subjects.

Description

[Field of the Invention]
The present invention relates to a human that cross-reacts with and / or blocks two or more inhibitory KIR receptors present on the cell surface of NK cells and enhances cytotoxicity of NK cells in a mammalian subject or biological sample. It relates to fragments and derivatives thereof as well as antibodies. The present invention relates to methods of making such antibodies, fragments, variants, and derivatives; pharmaceutical compositions comprising the same; and uses of such molecules and compositions, particularly NK cell activity or cells in a subject It also relates to their use in therapy to increase toxicity.

[Background of the invention]
Natural killer (NK) cells are a subpopulation of lymphocytes that are involved in immunity and in the host immune surveillance.

  NK cells are mononuclear cells that develop from lymphoid progenitor cells in the bone marrow, and morphological and biological characteristics typically include the following: cluster determinants (CDs) Expression of CD16, CD56, and / or CD57; absence of alpha / beta or gamma / delta TCR complexes on the cell surface; “self” major histocompatibility complex (MHC) / human leukocyte antigen ( The ability to bind and kill target cells that fail to express (HLA) protein; and the ability to kill tumor cells or other diseased cells that express a ligand to activate the NK receptor. NK cells are characterized by the ability to bind and kill several types of tumor cell lines without prior immunization or activation. NK cells can also release soluble proteins and cytokines that exert regulatory effects on the immune system; they also produce multiple rounds of cell division, producing daughter cells with similar biological properties to the parent cell can do. Upon activation by interferon and / or cytokines, NK cells require direct physical contact between NK cells and target cells for lysis of tumor cells and cells infected with intracellular pathogens It mediates by the mechanism. Target cell lysis involves releasing cytotoxic granules from NK cells to the surface of the bound target and effectors such as perforin and granzyme B that penetrate the target plasma membrane and induce apoptosis or programmed cell death Protein release is involved. Normal and healthy cells are protected from lysis by NK cells.

  Based on their biological properties, various therapeutic and vaccine strategies that rely on modulation of NK cells have been proposed in the art. However, NK cell activity is controlled by a complex mechanism involving both stimulatory and inhibitory signals.

  Briefly, the lytic activity of NK cells is controlled by various cell surface receptors that introduce either positive or negative intracellular signals upon interaction with ligands on target cells. The balance of positive and negative signals transmitted through their receptors determines whether target cells are lysed (killed) by NK cells. NK cell stimulating signals are NKp30, NKp44, and NKp46; as well as natural cytotoxic receptors (NCR; Natural Cytotoxicity Receptors) (Lanier, Annual Review of Immunology 2005; 23: 225-74). NK cell inhibitory signals can be mediated by receptors such as Ly49, CD94 / NKG2A (also specific inhibitory KIRs) that recognize major histocompatibility complex (MHC) class I molecules (Karre et al., Nature 1986; 319: 675-8; Ohlen et al, Science 1989; 246: 666-8). These inhibitory receptors bind to polymorphic determinants of MHC class I molecules (including HLA class I) present on other cells and inhibit NK cell-mediated lysis.

  KIRs, sometimes referred to as Killer Inhibitory Receptors, have been characterized in humans and non-human primates. The receptor is a polymorphic type 1 transmembrane molecule present on a specific subset of lymphocytes, including NK cells and some T cells. KIRs interact with determinants in the alpha 1 and 2 domains of MHC class I molecules. Also, as noted above, distinct KIRs are stimulatory or inhibitory to NK cells.

  The KIR nomenclature is the number of extracellular domains (KIR2D and KIR3D with 2 and 3 extracellular Ig domains, respectively) and the long cytoplasmic tail (KIR2DL or KIR3DL) or short (KIR2DS or KIR3DS) Based on how. The presence or absence of a given KIR varies from NK cell to NK cell within the NK population present in a single solid. Within the human population, the polymorphism level of the KIR gene is also relatively high, and certain KIR genes are present in some individuals but not in all individuals. The expression of KIR alleles in NK cells is stochastically controlled. That is, in a given individual, a given lymphocyte can express one, two or more different KIRs depending on the genotype of the individual. Single individual NK cells typically express different combinations of KIRs. This provides a repertoire of NK cells with different specificities for MHC class I molecules.

  Certain KIR gene products cause stimulation of lymphocyte activity when bound to the appropriate ligand. All activating KIRs have a short cytoplasmic tail with charged transmembrane residues associated with adapter molecules with immune receptor tyrosine-based activation motifs (ITAMs), thereby stimulating A signal is transmitted to NK cells. In contrast, inhibitory KIRs have a long cytoplasmic tail that contains an immunoreceptor tyrosine-based inhibition motif (ITIM). This transmits an inhibitory signal to NK cells upon association of their MHC class I ligands. Known inhibitory KIRs include members of the KIR2DL and KIR3DL subfamilies. Inhibitory KIRs having two Ig domains (KIR2DL) are HLA-C allotype: KIR2DL2 (previously labeled p58.2) and the closely related allele product KIR2DL3 1 ”HLA-C allotypes (including HLA-Cw1, -3, -7, and -8), while KIR2DL1 (p58.1) recognizes“ Group 2 ”HLA-C allotypes (eg, HLA -Cw2, -4, -5, and -6) are recognized. Recognition by KIR2DL1 is governed by the presence of a Lys residue located at position 80 of the HLA-C allele. Recognition of KIR2DL2 and KIR2DL3 is governed by the presence of the Asn residue located at position 80. Importantly, the majority of HLA-C alleles have either an Asn or Lys residue at position 80. Thus, KIR2DL1, -2, and -3 recognize collectively all HLA-C allotypes found fundamentally in humans. One of the KIDs with three Ig domains, KIR3DL1 (p70), recognizes an epitope shared by the HLA-Bw4 allele. Finally, KIR3DL2 (p140) (a homodimer of molecules with three Ig domains) recognizes HLA-A3 and -A11.

  Inhibitory KIR and / or other MHC class I specific inhibitory receptors (Moretta et al, Eur J Immunogenet. 1997; 24 (6): 455-68; Valiante et al, Immunol Rev 1997; 155: 155-64 Lanier, Annu Rev Immunol 1998; 16: 359-93) may be co-expressed by NK cells, but in any given individual's NK repertoire, there are cells that express only a single KIR. Thus, it is inhibited only by certain MHC class I alleles (or alleles belonging to the same group of MHC class I allotypes). Human MHC class I molecules are often referred to as human histocompatibility antigen (HLA) class I.

  NK cell populations or clones that are KIR-ligands that are mismatched with respect to their targets (ie, that express KIRs that do not recognize any HLA molecules in the host) can be obtained after allogeneic bone marrow transplantation in leukemia patients. Have been shown to mediate a powerful life-saving anti-tumor response (Ruggeri et al., Science 2002,295: 2097-2100). The underlying mechanism is believed to be: a donor-derived HLA-mismatched hematopoietic cell transplant expressing a KIR that does not recognize any HLA ligand in the recipient It induces the proliferation of NK cells and these are not inhibited through KIR. These allogenic NK clones exhibit potent antitumor activity. This response is very strong in patients diagnosed with acute myeloid leukemia (AML) and treated with a haplo-identical implant of the KIR-MHC mismatch. One way to reproduce this effect by the patient's pharmaceutical treatment would be to administer a reagent that blocks the KIR / HLA interaction to activate the patient's endogenous NK cells.

  Specific monoclonal antibodies specific for KIR2DL1 interact with KIR2DL1 and “Group 2” HLA-C allotypes [eg, HLA-Cw4 (Moretta et al., J Exp Med 1993; 178: 597-604)] It has been shown to block and promote NK-mediated lysis of target cells expressing these HLA-C allotypes. It has also been described that monoclonal antibodies against KIR2DL2 / 3 block the interaction of KIR2DL2 / 3 with HLA-Cw3 or similar allotypes (Moretta et al., J Exp Med 1993; 178: 597-604). Such antibodies are not ideal clinically. Because the development of two therapeutic monoclonal antibodies (mAbs) and the administration of both such antibodies or one choice of such antibodies (after appropriate diagnosis) to treat all potential patients As required, depending on whether any given patient was expressing group 1 or group 2 HLA-C allotypes.

  Watzl et al. (Tissue Antigens 2000; 56: 240-247) produced cross-reacting mouse antibodies that recognize multiple isotypes of KIR, but these antibodies enhance the lytic activity of NK cells. There wasn't. Furthermore, Spaggiari et al. (Blood 2002; 99: 1706-1714 and Blood 2002; 100: 4098-4107) conducted experiments using a number of mouse monoclonal antibodies against various KIRs. One of those antibodies, NKVSF1 (also known as Pan2D), was reported to recognize a common epitope of KIR2DL1 (CD158a), KIR2DL2 (CD158b) and KIR2DS4 (p50.3). Shin et al. (Hybridoma 1999; 18: 521-7) also reported the production of two monoclonal antibodies (referred to as A210 and A803g) that bind to all of KIR2DL1, KIR2DL3, and KIR2DS4. Have the ability. However, for therapeutic use of antibodies in blocking NK cell inhibitory KIRs in patients, it is better that fewer activated KIR molecules are cross-reacted with antibodies (since activated receptors Because it may impair the stimulation of NK cells). Thus, antibodies having antigen binding properties of NKVSF1, A210, or A803g may not be optimal in a clinical setting. Furthermore, the use of murine monoclonal antibodies in the treatment of human patients can lead to a host immune response against the antibody, thus compromising the effectiveness of the treatment.

  Thus, practical and effective approaches for therapeutic modulation of inhibitory KIRs have not previously been available in the art.

[Summary of Invention]
The present invention relates to novel and useful human antibodies that specifically bind to KIR2DL1 and at least one KIR2DL2 and KIR2DL3, or all three of these KIRs, and / or one or more such KIRs with NK-cytolytic activity. And human antibodies that enhance by blocking the interaction between HLA-C. Fragments and derivatives of such antibodies are also provided. The invention also includes novel and useful antibodies, antibody fragments, and antibody derivatives (which are substantially identical to those of human antibodies 1-7F9 and 1-4F1 described herein). Identical VH and VL sequences). The invention also includes a nucleic acid comprising a nucleotide sequence encoding such an antibody; a vector comprising such a nucleic acid; a host cell and organism comprising such a nucleic acid and / or vector; and a pharmaceutically acceptable A composition, such as a composition to be prepared, and such proteins, nucleic acids, vectors, and / or cells and typically one or more additional ingredients [this is the active ingredient or formulation, delivery of said composition, Kits are provided that may be inert components (eg, various carriers) that promote stability, or other characteristics. In addition, the present invention provides a variety of new and useful methods for making and using such antibodies, nucleic acids, vectors, cells, organisms and / or compositions (eg, in modulating KIR-mediated biological activity). E.g. in the treatment of diseases associated therewith).

  In one aspect, the invention provides a human or humanized antibody that binds to each one of KIR2DL1, KIR2DL2, and KIR2DL3, but does not bind to KIR2DS4. In one embodiment, the antibody does not further bind to KIR2DS3. In another embodiment, the human or humanized antibody blocks binding of at least one KIR2DL1, KIR2DL2, and KIR2DL3 to HLA-C class I molecules. In a further embodiment, the antibody may block the binding of HLA-Cw4 molecules to KIR2DL1 and the binding of HLA-Cw3 molecules to at least one KIR2DL2 or KIR2DL3. For example, the antibody can block binding of the HLA-Cw4 molecule to residues M44, F45, and D72 of the extracellular portion of KIR2DL1 (SEQ ID NO: 23). In yet another embodiment, the antibody enhances lytic activity of NK cells against human target cells expressing HLA-C class I molecules.

  In another aspect, the invention provides a light chain variable region comprising the amino acid sequence of SEQ ID NO: 15 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 17 in association with at least one KIR2DL1, KIR2DL2, and KIR2DL3. Human or humanized antibodies that compete with antibodies comprising variable regions are provided. In one aspect, the antibody comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 15 and a heavy chain variable comprising the amino acid sequence of SEQ ID NO: 17 in binding to at least one KIR2DL1, KIR2DL2, and KIR2DL3. Compete with an antibody comprising a region. In one aspect, the antibody comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 15. In yet another embodiment, the antibody comprises (a) an amino acid sequence of heavy chain CDR1 corresponding to residues 31 to 35 of SEQ ID NO: 17; (b) heavy chain CDR2 corresponding to residues 50 to 65 of SEQ ID NO: 17 And (c) the amino acid sequence of heavy chain CDR3 corresponding to residues 99-112 of SEQ ID NO: 17. For example, the antibody may comprise a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 17.

  In another aspect, the invention provides KIR2DL1 substantially comprising amino acid residues L38, R41, M44, F45, N46, D47, T48, L49, R50, I52, F64, D72, Y80, P87, and Y88. An isolated human or humanized antibody that binds to an epitope is provided.

In another aspect, the invention provides an isolated human or humanized antibody having a dissociation constant (K _d ) of about 0.45 nM or less for KIR2DL1 and / or a K _{d of} about 0.025 nM or less for KIR2DL3. provide.

The present invention also provides a human or humanized antibody or antibody fragment or derivative thereof having any of the aforementioned properties, alone or in any suitable combination. In one embodiment, the antibody is a monoclonal antibody. In another embodiment, the antibody is an IgG1, IgG2, IgG3, or IgG4 antibody. For example, the antibody may be an IgG4 antibody. The present invention also includes a nucleic acid encoding a human or humanized antibody or antibody fragment having any of the aforementioned properties, such nucleic acid. A method of producing a human anti-KIR antibody comprising culturing such a cell, cells containing such a vector, and such cells under conditions suitable for expression of the anti-KIR antibody.

  The present invention also provides a human or humanized antibody or antibody fragment having one or more of the aforementioned properties or produced by any method to the extent that NK cell cytotoxicity in a patient can be detected. A pharmaceutical composition comprising a pharmaceutically acceptable carrier or excipient in an effective amount. The composition includes, for example, polysorbate 80, sucrose, or both polysorbate 80 and sucrose.

  The present invention also provides a method of enhancing NK cell activity in a subject, the method comprising administering to said subject an effective amount of any of the aforementioned compositions. In one embodiment, the subject is a patient suffering from cancer. For example, the patient may have a cancer selected from acute myeloid leukemia, chronic myelogenous leukemia, multiple myeloma, and non-Hodgkin lymphoma. Alternatively, the patient may suffer from a cancer selected from colorectal cancer, kidney cancer, ovarian cancer, lung cancer, breast cancer, and malignant melanoma. In another embodiment, the subject is a patient suffering from an infection. In yet another embodiment, the method comprises an immunomodulator, a hormonal agent, a chemotherapeutic agent, an anti-angiogenic agent, an apoptotic agent, a second antibody that binds to an inhibitory KIR receptor, an anti-infective agent, a targeting inducer. further comprising administering a therapeutic agent selected from an agent, and an adjunct compound.

  The description of an antibody “provided” by the present invention or the description of an antibody such as “relates” (and by implication) to the present invention is clearly not contradicted unless stated otherwise or by context. To the extent it should be understood that it refers to an antibody that is useful in the practice of methods that meet the patent requirements described herein.

  These and other aspects and features of the invention are described in further detail herein.

[Definition]
For convenience, some terms are defined here. However, the defined list of terms provided herein is not exclusive. Other terms may be defined throughout the description of the invention.

  In the present invention, “active” NK cells refer to biologically active NK cells, which have the ability to lyse target cells or enhance the immune function of other cells. NK cells are included. For example, “active” NK cells can kill cells that express ligands for activated NK receptors and / or cannot express MHC / HLA antigens recognized by KIRs in NK cells. NK cells can be obtained by various techniques known in the art, such as isolation from a blood sample, leukocyte / granulocyte removal therapy, tissue, or cell collection. Useful protocols for assays involving NK cells can be found in the literature [Natural Killer Cells Protocols (edited by Campbell KS and Colonna M). Human Press. Pp. 219-238 (2000)].

As used herein, "Killer Ig-like Receptor", "Killer Inhibitory Receptor", or "KIR" is a gene that is a member of the KIR gene family Or a protein or polypeptide encoded by a cDNA prepared from such a gene. A detailed review of the KIR gene family (including KIR genes and KIR gene product nomenclature, and Genbank accession numbers for exemplary KIRs) is “The KIR Gene Cluster” by M. Carrington and P. Norman, It is available on the NCBI website called “Bookshelf” (connectable via World-Wide Web (WWW) address ncbi.nlm.nih.gov/books). The sequences of human KIR genes and cDNAs as well as their protein products are available in public databases including GenBank. Non-limiting Genbank entries for human KIRs have the following accession numbers: KIR2DL1: Genbank accession number U24076, NM_014218,, AAR16197, or L41267; KIR2DL2: Genbank accession number U24075 or L76669; KIR2DL3: Genbank accession Number U24074 or L41268; KIR2DL4: Genbank accession number X97229; KIR2DS1: Genbank accession number X89892; KIR2DS2: Genbank accession number L76667; KIR2DS3: Genbank accession number NM_012312 or L76670 (splice variant); KIR3DL1: Genbank accession number L41269 And KIR2DS4: Genbank accession number AAR26325. A KIR may comprise 1-3 extracellular domains and may have a long (ie, greater than 41 amino acids) or short (ie, less than 40 amino acids) cytoplasmic tail. As described herein, these properties determine the KIR nomenclature. Exemplary KIR2DL1, KIR2DL2, KIR2DL3, and KIR2DS4 molecules have the respective amino acid sequences described below:
KIR2DL1 extracellular domain:
EichiijibuieichiarukeiPiesueruerueieichipijiEkkusuerubuikeiesuiitibuiaierukyushidaburyuesudibuiemuefuieichiefueruerueichiaruijiemuefuenuditieruarueruaijiieichieichidijibuiesukeieienuefuesuaiesuaruemutikyudierueijitiwaiarushiwaijiesubuitieichiesuPiwaikyubuiesueiPiesudiPierudiaibuiaiaijieruwaiikeiPiesueruesueikyuEkkusuJipitibuierueijiienubuitieruesushiesuesuaruesuesuwaidiemuwaieichieruesuaruijiieieichiiaruarueruPieiJipikeibuienujitiefukyueidiefuPieruJipieitieichijijitiwaiarushiefujiesuefueichidiesuPiwaiidaburyuesukeiesuesudiPieruerubuiesubuiTGNPSNSWPSPTEPSSKTGNPRHLH (SEQ ID NO: 23), "X" at position 16 in the sequence is P or R, at position 114 "X" is P or L, representing allelic variants.

KIR2DL2 extracellular domain:
HEGVHRKPSLLAHPGRLVKSEETVILQCWSDVRFEHFLLHREGKFKDTLHLIGEHHDGVSKANFSIGPMMQDLAGTYRCYGSVTHSPYQLSAPSDPLDIVITGLYEKPSLSAQPGPTVLAGESVTLSCSSRSSYDMYHLSREGEAHECRFSAGPKSVNGSNSQ
KIR2DL3 extracellular domain:
HEGVHRKPSLLAHPGPLVKSEETVILQCWSDVRFQHFLLHREGKFKDTLHLIGEHHDGVSKANFSIGPMMQDLAGTYRCYGSVTHSPYQLSAPSDPLDIVITGLYEKPSLSAQPGPTVLAGESVTLSCSSRSSYDMYHLSREGEAHERRFSAGPKVNGTGTFQADFPLGPATHG

KIR2DS4 extracellular domain:
QEGVHRKPSFLALPGHLVKSEETVILQCWSDVMFEHFLLHREGKFNNTLHLIGEHHDGVSKANFSIGPMMPVLAGTYRCYGSVPHSPYQLSAPSDPLDMV (SEQ ID NO: 38).

  The term “KIR2DL2 / 3” refers to one or both of the KIR2DL2 and KIR2DL3 receptors. These two receptors have very high homology, are allelic forms of the same gene, and are considered functionally similar by the art.

  Unless otherwise specified, the term “MHC” includes MHC molecules in all mammals. On the other hand, an “HLA” molecule means a human MHC molecule.

  In the present invention, the description that an antibody “binds” to a determinant (ie, the term “bind”: determinant interaction in the context of an antibody) means that the antibody is determined with specificity and / or affinity. It means to bond to a group. For example, GL183 is a conventional monoclonal antibody that binds to KIR2DL2 / 3. EB6 is a conventional monoclonal antibody that binds to KIR2DL1. EB6 and GL183 are both commercially available (Beckman Coulter Inc., Fullerton, CA).

  A “cross-reactive” anti-KIR antibody is an antibody that binds with specificity and / or affinity to two or more KIR molecules. For example, DF200 and 1-7F9 are monoclonal antibodies that are cross-reactive with KIR2DL1, -2, and -3. The hybridoma producing the antibody DF200 was identified with the identification number “June 10, 2004 registered in“ Collection Nationale de Cultures de Microorganismes, Institut Pasteur, 25, Rue du Docteur Roux, F-75724 Paris Cedex 15, France ””. It was deposited with the CNCM culture collection as “DF200” and registration number CNCM “CNCM I-3224”. NKVSF1 (also referred to herein as “Pan2D”) is cross-reactive with KIR2DL1, -2, and -3 and KIR2DS4. This antibody is commercially available from Serotec (Cergy Sainte-Christophe, France) under catalog number MCA2243.

  "Specific binding" or "specificity" binds detectably to an epitope presented on an antigen (eg, KIR), but other proteins or structures (eg, By other proteins presented on NK cells or other cell types) is meant the ability of an antibody or other agent to have a relatively low detectable reactivity. Specificity can be determined relatively by binding or competitive binding assays, such as with a Biacore instrument described elsewhere herein. Specificity is about 10: 1, about 20: 1, about 50: 1, about 100: 1 of the affinity / binding activity of binding to a specific antigen versus nonspecific binding to other unrelated molecules. , 10.000: 1 or higher (in this case, the specific antigen is KIR). KIR-binding antibodies (specific for KIR displayed on NK cells of certain organisms such as humans) may occasionally show binding to similar KIRs of other species (ie KIR-binding) Antibodies, or other KIR-binding agents, cross-react with various species of KIRs).

  “Selectivity” means that a protein preferentially binds to a particular region, target, or peptide rather than one or more other biological molecules, structures, cells, tissues, and the like. Alternatively, KIR binding antibodies may be selected for KIR produced in a particular organism (eg, in primates versus in humans) and / or for certain types of KIRs (eg, KIRs with long cytoplasmic tails) In particular, the antibody may cross-react with more than one type of KIR produced in a particular organism and / or with a particular portion of the KIR (eg, a particular epitope or antigenic determinant region). For example, selectivity can be determined by competitive ELISA or Biacore assays. The difference in affinity / binding activity representing selectivity may be of any detectable priority (eg, a ratio greater than 1: 1.1 or greater than about 1: 5 is appropriate if detectable). Well, this includes 1:10, 1: 100, 1: 1000 and more). Unless otherwise specified, any quantitative data in “affinity” described herein means a measure of bivalent (not monovalent) binding.

  “Epitopes” or “binding sites” are areas or regions on an antigen to which an antigen-binding peptide (eg, antibody) specifically binds. Protein epitopes are those that bind to amino acid residues that are directly involved in binding (also referred to as the immunodominant component of the epitope) and amino acid residues that are efficiently blocked by specific antigen-binding peptides. Other amino acid residues that are not directly involved (in other words, the amino acid residues are within the “footprint” of the specific antigen-binding peptide) may be included. Unless otherwise stated, the term epitope herein includes both types in any particular region of KIR that specifically binds to an anti-KIR antibody or another KIR-specific factor according to the present invention. (Eg, in some contexts, the invention relates to antibodies that bind directly to specific amino acid residues). KIRs may include several different epitopes, including but not limited to (1) linear peptide antigenic determinants and (2) localities close to each other in the mature KIR conformation. Any conformational antigenic determinant consisting of one or more non-contiguous amino acids present; and (3) either all or part of the molecular structure (eg, sugar group) covalently attached to the KIR It may contain an antigenic determinant after translation modification consisting of these.

  The expression that the first antibody binds “substantially” or “at least partially” to the same epitope as the second antibody means the following; The epitope binding site for the first antibody is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80 on the antigen constituting the epitope binding site of the second antibody %, 90%, or more amino acid residues. In addition, the fact that the first antibody substantially or partially binds to the same epitope as the second antibody means that the first and second antibodies compete for binding to the antigen as described above. To do. Thus, the term “binds to substantially the same epitope or determinant as” with monoclonal antibody 1-7F9 means that the antibody “competes” with 1-7F9. Means. In general, a subject monoclonal (eg, DF200, NKVSF1, 1-7F9) antibody and an antibody that “binds substantially the same epitope or determinant” comprise one or more KIR molecules (preferably KIR2DL1 and KIR2DL2 / Means to “compete” with the antibody of interest for binding to a KIR molecule selected from the group consisting of 3. In another example, an antibody that binds substantially the same epitope or determinant on the KIR2DL1 molecule as the antibody of interest “competes” with the antibody of interest for binding to KIR2DL1. An antibody that binds to an epitope or determinant on the KIR2DL2 / 3 molecule that is substantially the same as the antibody of interest "competes" with the antibody of interest for binding to KIR2DL2 / 3.

  The expression “binds to substantially the same epitope or determinant” as an antibody of interest refers to at least one or any and all KIR molecules to which the antibody of interest specifically binds that one antibody of the subject. It means “competing” with an antibody. The expression "binds to substantially the same epitope or determinant" as monoclonal antibody 1-7F9 is an antibody that relates to at least one, preferably any and all KIR molecules, that 1-7F9 specifically binds Means “compete” with 1-7F9. For example, an antibody that binds to essentially the same epitope or determinant as monoclonal antibody 1-7F9 or NKVSF1 "competes" with 1-7F9 or NKVSF1 for binding to KIR2DL1, KIR2DL2 / 3, KIR2DS1, and KIR2DS2, respectively. "be able to.

  The ability of an anti-KIR antibody to “block” the binding of a KIR molecule and an HLA molecule means the following: the antibody may be soluble or cell surface associated KIR and HLA molecules In the assay used, it is possible to detectably reduce the binding of the KIR molecule to the HLA molecule in a dose-dependent manner (where the KIR molecule is the absence of the HLA molecule and the antibody Bind detectably below). An exemplary assay for determining whether an anti-KIR antibody is capable of such blocking is provided in Example 8.

  Anti-KIR antibodies “reduce the inhibitory activity of a KIR”, “facilitate NK cell activity”, “facilitate NK cell cytotoxicity (facilitate NK cell cytotoxicity ”,“ facilitate NK cells ”,“ potentiate NK cell activity ”,“ potentiate NK cell cytotoxicity ” Or the ability to “potentiate NK cells” means the following: NK cells expressing KIR when contacted with said antibody are It has the ability to lyse target cells that express a particular MHC or HLA class I (which is a ligand for said KIR) on the surface. For example, the term “potentiation in NK cytotoxicity” refers to any substantial enhancement or at least 5%, 10%, 20%, 30% or more NK cytotoxicity compared to a control. Enhancement, eg, at least about 50% enhancement of target NK cytotoxicity (eg, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% Refers to, for example, about 55-100%, about 65-100%, about 75-100%, about 80-100%, or about 90% of NK cell cytotoxicity as compared to a control. This includes ~ 100% enhancement, and includes cytotoxicity increases of about 100% or more, about 500% or more, about 1000% or more, about 2000% or more, such as standard cytotoxicity assays. In this assay, a number of Target cells high percentage reaches) is, NK potentiator (e.g., in the presence of anti-KIR mAb), the absence of the potentiator (i.e., control) are lysed by NK cells than.

  As used herein, the term “neutralize KIR-mediated inhibition of NK cell cytotoxicity” or “neutralize the inhibitory activity of KIR” is a traditional chromium release test for cytotoxicity. About 20% or more, preferably at least about 30%, at least about 40% of the specific lysis obtained at the same effector / target cell ratio as NK cells or NK cell lines that are not blocked by their KIR as measured. %, The ability to increase to at least about 50%, at least about 100% or more. “Neutralize KIR-mediated inhibition” also means the following: on NK cell clones or on transfectants and NK cells expressing one or more inhibitory KIRs In a chromium release assay or other cytotoxicity assay using target cells expressing at least one HLA class I allele recognized by one of the KIRs, the specific lysis obtained with said antibody is About 100% or more, preferably at least about 101%, at least about 150, obtained with the same effector: target cell ratio at the same concentration of NKVSF1 (commercially available from Serotec). %, At least about 200% or more (e.g., about 101-150%, about 120-150%, about 120-200%, about 150-200%, or about 200-1000%), or more specific lysis It is to be.

  The term “human antibody” as used herein refers to or is essentially identical to or derived from a human germline immunoglobulin sequence. It is intended to include antibodies having variable and constant regions. Such human antibodies can comprise amino acid residues that are not encoded by human germline immunoglobulin sequences (eg, by performing random or site-directed mutagenesis in vitro or by performing somatic mutations in vivo). Mutation introduced by). However, as used herein, the term “human antibody” includes antibodies in which CDR sequences derived from the germline of other mammalian species (eg, mice) are grafted onto human framework sequences. Not intended.

  In the present invention, unless stated otherwise, the term “treatment” or “treating” means one or more symptoms of a disease or disorder, unless otherwise contradicted by context. Means preventing, preventing, alleviating, managing, curing, or reducing symptoms or clinically relevant manifestations. For example, “treatment” of a patient for whom no symptom or clinically relevant sign of the disease or disorder has been identified is a prophylactic therapy, but for a patient for whom a symptom or clinically relevant sign of the disease or disorder has been identified. “Treatment” generally does not constitute a preventive therapy. In contrast, the following should be understood: from which the various therapeutic and prophylactic methods and use facets of the present invention are distinguished in many ways [eg, The dose of compound to be delivered to the subject, the timing of application, the impetus of application, etc.] each can be considered a unique aspect of the present invention.

  In the present invention, “cancer” means any neoplastic disorder, including cellular disorders such as sarcoma, carcinoma, melanoma, leukemia, and lymphoma, breast, head And head and neck, ovary, bladder, lung, pharynx, larynx, esophagus, stomach, small intestine, liver, pancreas, colon, female reproductive tract, male reproductive tract, prostate, kidney, And cancers in the central nervous system, including but not limited to.

  The term “biological sample” includes biological fluids (eg, serum, lymph, and blood), cell samples or tissue samples (eg, bone marrow or tumor tissue) derived from a human or non-human mammal. However, it is not limited to these.

  The term “substantially identical” in the context of two amino acid sequences means the following: such as by using a default gap weight in the program GAP or BESTFIT When optimally aligned, the sequences are at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, at least about 95, at least about 98, or at least about 99 percent sequence identity ( sequence identity). In one aspect, residue positions that are not identical differ by conservative amino acid substitutions (described further elsewhere herein). Sequence identity is typically measured using sequence analysis software. By protein analysis software, similar sequences were matched using similarity criteria and assigned to various substitutions, deletions, and other modifications, including conservative amino acid substitutions. As an example, publicly available GCG software includes programs such as “Gap” and “BestFit”, which use default parameters to establish a close relationship between closely related polypeptides (eg, different species of organisms). Sequence homology) or wild type protein and its mutant protein (mutein) can be determined. See GCG version 6.1. Polypeptide sequences can also be compared using FASTA and applying default or recommended parameters. The program in GCG version 6.1, FASTA (eg, FASTA2 and FASTA3), provides optimal overlapping region alignment and percent sequence identity between query and search sequences (Pearson, Methods Enzymol. 1990; 183 : 63-98; Pearson, Methods Mol. Biol. 2000; 132: 185-219). Another suitable algorithm for comparing a particular sequence to a database containing many sequences from different organisms is the computer program BLAST (especially using blastp with default parameters). For example, Altschul et al. [Altschul et al., J. Mol. Biol. 1990; 215: 403-410; Altschul et al., Nucleic Acids Res. 1997; 25: 3389-402 (1997)]; Incorporated by reference in the text. “Corresponding” amino acid positions in two substantially identical amino acid sequences are alignments of any of the protein analysis software referred to herein using default parameters.

  As used herein, a “1-7F9-like” or “1-4F1-like” antibody comprises (1) 1-7F9 or 1-4F1 VH and VL sequences, respectively. Antibodies that bind to substantially the same epitope as the existing antibody (eg, monoclonal antibodies 1-7F9 or 1-4F1 respectively) and / or (2) identical or substantially identical to the VH and VL sequences of 1-7F9 or 1-4F1 respectively Antibodies that contain identical VH and VL sequences.

  A “conservative” amino acid substitution is another amino acid residue in which an amino acid residue has a side chain (“R-group”) with similar chemical properties (eg, charged or hydrophobic). Substituted by a group. Usually, conservative amino acid substitutions do not substantially change the functional properties of the protein. Where amino acid sequences differ from each other by conservative substitutions, the percent sequence identity may be adjusted upwards and corrected for the conservative nature of the substitutions. Means for making this adjustment are well known to those skilled in the art. See, for example, Pearson (Pearson, Methods Mol. Biol. 1994; 243: 307-31). Examples of groups of amino acids with side chains with similar chemical properties include 1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; 2) aliphatic hydroxyl side chains: serine and threonine ; 3) Amide-containing side chains: asparagine and glutamine; 4) Aromatic side chains: phenylalanine, tyrosine, and tryptophan; 5) Basic side chains: lysine, arginine, and histidine; 6) Acidic side chains: asparagine Acids and glutamic acids; and 7) sulfur-containing side chains: including cysteine and methionine. Exemplary conservative amino acid substituents include: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic acid-aspartic acid, and asparagine-glutamine.

[Description of the invention]
The present invention is based on the production of novel cross-reactive neutralizing antibodies that bind to inhibitory KIRs, which are effective in the activity of NK cells in most or all individuals in the human population. Tolerate

  What is described herein, for example, enhances the lytic activity of NK cells by binding to all of KIR2DL1, KIR2DL2, and KIR2DL3 and blocking the interaction between these KIRs and HLA-C Antibody. Such antibodies are referred to herein as “cross-reactive and neutralizing anti-KIR mAbs”.

  In certain aspects, the present invention provides novel cross-reactive, neutralizing, or cross-reactive and neutralizing globally human anti-KIR antibodies, as well as compositions comprising such antibodies And methods using such antibodies or compositions. These antibodies are human antibodies 1-7F9 and 1-4F1, and are described in PCT / DK2004 / 000470 filed on July 1, 2004, which is incorporated herein by reference. The whole is incorporated).

  The antibodies, compositions, and methods described herein overcome and add to the current limitations associated with, inter alia, the therapeutic modulation of KIR and with the activation of therapeutic NK cells. Can provide advantageous properties and benefits. For example, the antibodies can cross-react with multiple inhibitory KIR groups and reduce or neutralize their inhibitory signals, thereby causing NK cells expressing such inhibitory KIR receptors. Enhances cytotoxicity of NK cells. The ability to cross-react with multiple KIR gene products to efficiently use the antibodies of the present invention to predetermine NK cell activity in most or all human subjects, the KIR or HLA type of the subject Or it is acceptable to increase the cost without doing so. In one embodiment, the antibody does not bind to KIR2DS4, thus avoiding a decrease in stimulatory potential that would be associated with neutralization of activated KIR2DS4 receptor. Additionally or alternatively, the antibody does not bind to KIR2DS3.

  Various antibody fragments and derivatives can also be produced from such antibodies and used for the same or similar purposes as described herein for the antibodies of the invention. Also, the aspects of the invention described in connection with antibodies apply equally to antibody fragments and derivatives unless otherwise stated or unless clearly contradicted by context. In other words, the features of the invention described herein in connection with antibodies relate to antibody fragments or derivatives that have similar functionality in terms of specificity unless otherwise stated. Thus, describing similar features should be understood implicitly. However, it should be recognized that: “full-length” antibodies and antibody “fragments” differ significantly in their biological and physicochemical properties. As long as it is characterized as a separate aspect of the present invention.

  Some antibodies of the invention are characterized as “human”. Typically this has a low risk for an immune response against said antibodies by the human subject to whom they are administered. In one exemplary aspect, an isolated antibody that promotes activation of human NK cells in virtually all humans is described. In typical embodiments, the antibody is a human antibody 1-7F9 or a human 1-7F9-like antibody.

  The antibody, fragment, or derivative of any of them cross-reacts with at least two inhibitory KIR receptors on the surface of the NK cell, reduces or neutralizes the inhibitory signal of the NK cell, and The activity can be enhanced. For example, if an antibody, antibody fragment, or derivative can bind to a common determinant of the human KIR2DL receptor, the antibody, antibody fragment, or derivative binds at least to the KIR2DL1, KIR2DL2, and KIR2DL3 receptors. In the present invention, the term “KIR2DL2 / 3” refers to one or both of the KIR2DL2 and KIR2DL3 receptors.

As described herein, anti-KIR mAbs 1-7F9 and 1-4F1 have several advantages over previously generated anti-KIR antibodies. For example, since 1-7F9 and 1-4F1 are fully human, any immune response to the antibody once administered to the subject is reduced or minimized. Furthermore, both 1-7F9 and 1-4F1 are of the appropriate isotype for therapeutic anti-KIR antibodies as described below (IgG4 and IgG2, respectively). In addition, 1-7F9 is more effective than murine mAbs EB6, GL183, DF200, and NKVSF1 (Pan2D) in inducing killing by NK cells that express either KIR2DL1, -2, and / or -3 It is. For example, as described in FIGS. 5 and 6, 1-7F9 produces a higher level of specific lysis of target cells expressing HLA-Cw4 by KIR2DL1-expressing NK cells than by EB6, DF200 or NKVSF1 (Pan2D). Induce. 1-7F9 has a higher affinity for KIR compared to the previously known anti-KIR mAbs. For example, 1-7F9 binds to KIR2DL1 and KIR2DL3 with dissociation constants (K _d 's) of 0,43 nM and 0.025 nM, respectively. This result shows a higher affinity for both antibodies than DF200 etc. (see Examples 3 and 8). In contrast to mouse antibodies NKVSF1 (Pan2D), A210, and A208g, 1-7F9 and 1-4F1 do not bind to KIR2DS4, making them more suitable for therapeutic purposes. Like NKVSF1 (Pan2D) and DF200, 1-7F9 and 1-4F1 also bind to KIR2DS1 and KIR2DS2, but KIR2DS1 and KIR2DS2 are not believed to be important for anti-leukemic efficacy. Certain antibodies according to the invention have the same or similar antigen specificity as 1-7F9 and / or 1-4F1. For example, an antibody comprising the same or similar VH and VL regions as 1-7F9 can have the same or similar antigen binding and / or NK stimulation properties as 1-7F9; and the same as 1-4F1 Alternatively, an antibody comprising similar VH and VL regions can have the same or similar antigen binding properties as 1-4F1.

As shown in FIG. 14, the amino acid sequences of the VL and VH regions of 1-7F9 were determined:
1-7F9 VL region (SEQ ID NO: 15):
EIVLTQSPVTLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWMYTFGQGTKLEIKRT
1-7F9 VH region (SEQ ID NO: 17):
QVQLVQSGAEVKKPGSSVKVSCKASGGTFSFYAISWVRQAPGQGLEWMGGFIPIFGAANYAQKFQGRVTITADESTSTAYMELSSLRSDDTAVYYCARIPSGSYYYDYDMDVWGQGTTVTVSS
The amino acid sequences of the 1-4F1 VL and VH regions are provided in SEQ ID NOs: 39 and 41, respectively. Nucleotide sequences encoding 1-4F1 VL and VH regions are also provided in SEQ ID NOs: 40 and 42, respectively. In certain embodiments, residues 3, 4, 9, 24, 32, 41, 47, 50, 55, 71, and 74 of SEQ ID NO: 39 are respectively Q, L, S, R, A, G, L, D, E, F, and A. In another specific embodiment, residues 3, 4, 9, 24, 32, 41, 47, 50, 55, 71, and 74 of SEQ ID NO: 39 are R, M, F, W, Y, A, F, Y, Q, Y, and T.

  As shown in FIG. 15, the amino acid sequence of 1-7F9 CDRs was identified as follows: light chain CDR1 amino acid sequence corresponds to residues 24-34 of SEQ ID NO: 15; light chain CDR2 amino acid The sequence corresponds to residues 50-56 of SEQ ID NO: 15; the light chain CDR3 amino acid sequence corresponds to residues 89-97 of SEQ ID NO: 15; the heavy chain CDR1 amino acid sequence is residues 31 of SEQ ID NO: 17 The heavy chain CDR2 amino acid sequence corresponds to residues 50-65 of SEQ ID NO: 17; and the heavy chain CDR3 amino acid sequence corresponds to residues 99-112 of SEQ ID NO: 17. The amino acid sequence of 1-4F1 CDRs was identified as follows: the light chain CDR1 amino acid sequence corresponds to residues 24-34 of SEQ ID NO: 39; the light chain CDR2 amino acid sequence is of SEQ ID NO: 39 The light chain CDR3 amino acid sequence corresponds to residues 89-97 of SEQ ID NO: 39; the heavy chain CDR1 amino acid sequence corresponds to residues 31-35 of SEQ ID NO: 41; heavy The chain CDR2 amino acid sequence corresponds to residues 50-66 of SEQ ID NO: 41; and the heavy chain CDR3 amino acid sequence corresponds to residues 99-113 of SEQ ID NO: 41.

  The amino acid sequences for the entire light and heavy chains of 1-7F9 are provided in SEQ ID NOs: 36 and 37, respectively.

Thus, for example, additional antibodies of various human antibody subclasses; antibody fragments, antibody derivatives, and other KIR binding peptides can be readily generated based on this information, such as by recombinant techniques. For example, in one aspect, the invention consists essentially of an antibody having VL and VH sequences consisting essentially of SEQ ID NO: 15 and SEQ ID NO: 17, respectively, and / or essentially SEQ ID NO: 39 and SEQ ID NO: 41, respectively. An antibody having the VL and VH sequences is provided. In another aspect, the present invention provides an antibody comprising a CDR region consisting essentially of the above 1-7F9 or 1-4F1 VH CDR1-3 and VL CDR1-3. In another aspect, the present invention provides an antibody comprising the following CDR regions: light chain CDR1 amino acid sequence corresponding to residues 24-34 of SEQ ID NO: 15; residues of SEQ ID NO: 15 Light chain CDR2 amino acid sequence corresponding to approximately 50-56; light chain CDR3 amino acid sequence corresponding to residues approximately 89-97 of SEQ ID NO: 15; corresponding to residues 31-35 of SEQ ID NO: 17 A heavy chain CDR1 amino acid sequence corresponding to residues about 50-65 of SEQ ID NO: 17; a heavy chain CDR3 amino acid sequence corresponding to residues about 99-112 of SEQ ID NO: 17. In another aspect, the present invention provides an antibody comprising the following CDR regions: a light chain CDR1 amino acid sequence corresponding to residues 24-34 of SEQ ID NO: 39; residues of SEQ ID NO: 39 Light chain CDR2 amino acid sequence corresponding to approximately 50-56; light chain CDR3 amino acid sequence corresponding to residues approximately 89-97 of SEQ ID NO: 39; corresponding to residues 31-35 of SEQ ID NO: 41 A heavy chain CDR1 amino acid sequence corresponding to residues 50-66 of SEQ ID NO: 41; a heavy chain CDR3 amino acid sequence corresponding to residues 99-113 of SEQ ID NO: 41. In another aspect, the present invention provides an antibody comprising the following amino acid sequence: light chain CDR1 amino acid sequence consisting essentially of residues 24-34 of SEQ ID NO: 15; residue 50 of SEQ ID NO: 15 A light chain CDR2 amino acid sequence consisting essentially of residues 89 to 97 of SEQ ID NO: 15; a heavy chain CDR1 consisting essentially of residues 31 to 35 of SEQ ID NO: 17 Amino acid sequence; residues 50-6 of SEQ ID NO: 17
A heavy chain CDR2 amino acid sequence consisting essentially of 5; a heavy chain CDR3 amino acid sequence consisting essentially of residues 99-112 of SEQ ID NO: 17. In another aspect, the invention provides an antibody comprising the following CDR regions: a light chain CDR1 amino acid sequence consisting essentially of residues 24-34 of SEQ ID NO: 39; residue 50 of SEQ ID NO: 39 A light chain CDR2 amino acid sequence consisting essentially of residues 89 to 97 of SEQ ID NO: 39; a heavy chain CDR1 consisting essentially of residues 31 to 35 of SEQ ID NO: 41 A heavy chain CDR2 amino acid sequence consisting essentially of residues 50 to 66 of SEQ ID NO: 41; a heavy chain CDR3 amino acid sequence consisting essentially of residues 99 to 113 of SEQ ID NO: 41.

  The present invention provides an anti-KIR antibody, antibody fragment, or antibody fragment comprising at least one variant amino acid sequence substantially identical to a VH or VL sequence of 1-7F9 or 1-4F1 or a CDR-region therein, or Also encompassed are antibody derivatives, or KIR binding polypeptides. The variant amino acid sequence has a CDR, VH, or VL region of 1-7F9 or 1-4F1 and at least about 50, 80, 90, 95, 98, or 99 (e.g., about 50-99, about 65-99, About 75-99, or about 85-99) percent identical amino acid sequences. The variant amino acid sequence comprises 1, 2, or 3 CDRs comprising or consisting of an amino acid sequence that is at least about 80%, at least about 90%, or at least about 95% identical to CDRs of 1-7F9 or 1-4F1 May also be included (also) or alternatively. Thus, in one aspect, the invention provides a light chain CDR1 amino acid sequence that is at least about 80%, at least about 90%, or at least about 95% identical to residues 24-34 of SEQ ID NO: 15 or SEQ ID NO: 39; A light chain CDR2 amino acid sequence that is at least about 80%, at least about 90%, or at least about 95% identical to residues 50-56 of SEQ ID NO: 15 or SEQ ID NO: 39; residues 89-97 of SEQ ID NO: 15 or SEQ ID NO: 39 A light chain CDR3 amino acid sequence that is at least about 80%, at least about 90%, or at least about 95% identical to; and at least about 80%, at least about 90%, or residues 31-35 of SEQ ID NO: 17 or SEQ ID NO: 41, or Heavy chain CDR1 amino acid sequence that is at least about 95% identical; at least about 80%, at least about 90%, or at least about 95% identical to residues 50-65 of SEQ ID NO: 17 or residues 50-66 of SEQ ID NO: 41 A heavy chain CDR2 amino acid sequence of SEQ ID NO: 17 with residues 99-112 or It provides human antibodies that are equipped with; residues 99-113 and at least about 80% of the number 41, at least about 90%, or at least about heavy chain CDR3 amino acid sequence is 95% identical. Basic characteristics of 1-7F9- or 1-4F1-derived KIR-binding amino acid sequences retained in the amino acid sequence of such variants (1-7F9- or 1-4F1-derived KIR-binding amino acid sequences) Desirably, the specificity and / or avidity of 1-7F9 or 1-4F1 sequences for one or more KIRs is included. The property may also include, alternatively or alternatively, the ability of 1-7F9 in blocking KIR / HLA-C interaction and enhancing lytic activity of NK cells.

  In another aspect, the invention provides one or more amino acid residues (e.g., at least 2, 3, 5, at least about 10, at least about 15, at least about 20, from a 1-7F9 or 1-4F1 KIR binding sequence). At least about 25, at least about 30, at least about 35, at least about 40, at least about 50 or more amino acid residues) depending on the mode of insertion, deletion and / or substitution of one or more residues An anti-KIR antibody, antibody fragment, or antibody derivative, or KIR-binding polypeptide comprising a sequence is provided. In one embodiment, such variant KIR binding sequences have high affinity; high or different specificity; low immunogenicity (in terms of host response to said sequence); high in vivo stability; and / or native 1 Other beneficial properties are imparted to variant sequences for essentially the same amino acid sequence comprising a -7F9 or 1-4F1 sequence. Suitable sequence variations are further described elsewhere herein. An anti-KIR antibody, antibody fragment, or antibody derivative, or KIR-binding portion of a KIR-binding polypeptide, promotes KIR binding and / or provides other advantageous physicochemical or immunological properties. Any number of non-amino acid components or substituents (eg, non-amino acid organic components) may also be included.

Some antibodies of the invention may be further or alternatively characterized by their binding affinity for one or more KIRs. For example, as shown in Examples 3 and 8, with respect to divalent bonding, DF200 is K _d for about 11nM respect KIR2DL1, and the K _d for about 2.0nM respect KIR2DL3, and 1-7F9 are of approximately 0.43 nM with respect to KIR2DL1 K _d And a K _d of about 0,025 nM for KIR2DL3. Accordingly, in one aspect, the present invention has a K _d of divalent bonds to KIR2DL1 of about 20 nM or less, about 11 nM or less, about 5 nM or less, about 1 nM or less, about 0.5 nM or less, about 0.43 nM or less. Human or non-human (eg, murine, chimeric, or humanized) antibodies are provided. Additionally or alternatively, a human or non-human (eg, murine, chimeric, or humanized) antibody of the invention has about 20 nM or less, about 2 nM or less, about 1 nM or less, about 0.1 nM or less with respect to KIR2DL3, or It may have a K _{d of} about 0.05 nM or less, or about 0.025 nM or less. In a particular aspect, the antibody has a K _d value that is approximately the same as 1-7F9 for divalent binding to KIR2DL1 and KIR2DL3. As shown in Example 13, for monovalent binding, 1-7F9 and 1-4F1 have K _d -values of about 3.5 and 7 nM for KIR2DL3, respectively. Accordingly, in one aspect, the invention provides a human or non-human (eg, mouse) monovalent binding to KIR2DL3 having a K _d of about 20 nM or less, about 10 nM or less, about 7 nM or less, about 3.5 nM or less. (Chimeric, or humanized) antibodies are provided.

  An anti-KIR antibody, antibody fragment, or antibody derivative, or KIR-binding polypeptide may be selectively and / or specifically (typically specifically) at least one KIR (more specifically at least 1 Two KIR antigenic determinants or epitopes) under suitable conditions (eg, in normal or NK cell-related disease states and in the context of suitable proteins containing said sequences or combinations) In terms of temperature, pH, etc.). For example, in one aspect, the invention relates to antibodies that can be characterized (among other things) in their ability to compete with 1-7F9, 1-4F1, or 1-7F9- or 1-4F1-like antibodies, and various involving the same Related to different methods. Other antibodies of the invention (and / or those useful in practicing the inventive methods described herein) compete with one or more of antibody DF200, antibody NKVSF1, antibody EB6, and antibody GL183. Having the capability can be further or alternatively characterized.

  Cross-reactive and neutralizing anti-KIR antibodies, antibody fragments, or derivatives of the present invention specifically inhibit the inhibitory activity of KIR and bind MHC and / or HLA molecules to at least two inhibitory KIR receptors. It is reduced or neutralized by inhibiting and promoting NK cell activity. This matter allows such antibodies, fragments, or derivatives to express inhibitory KIR on the surface of NK cells, and corresponding HLA ligands (eg, specific It means acquiring the ability to lyse cells expressing the HLA antigen. In one aspect, the present invention provides antibodies that specifically inhibit the binding of HLA-C molecules to KIR2DL1 and KIR2DL2 / 3 receptors. In another aspect, the present invention provides an antibody that inhibits KIR2DL1 and / or KIR2DL2 / 3 from binding to HLA-C. In yet another aspect, the present invention provides antibodies that promote NK cell activity in vivo and / or in vitro.

  At least one KIR2DL1 or KID2DL2 / 3 is present in at least about 90% or more of the human population. A more preferred antibody of the present invention also has the ability to enhance the activity of NK cells expressing either or both of these KIRs. Thus, the compositions of the present invention can be used to effectively activate or enhance NK cells in most human individuals, typically in about 90% or more human individuals. Thus, a single antibody composition according to the present invention can be used to treat most human subjects to determine KIR- or HLA-allele groups or a mixture of two or more anti-KIR mAbs or You rarely need to use a cocktail.

  In one aspect, the antibody specifically binds to both KIR2DL1 and KIR2DL2 / 3 human receptors and reverses the inhibition of NK cell cytotoxicity mediated by these KIRs. The antibody may be human and may compete with monoclonal antibodies 1-7F9 and / or 1-4F1. The term “competes with” refers to a specific pair of antibodies (eg, one or more antibodies selected from DF200, NKVSF1 (Pan2D), 1-7F9, EB6, and GL183). Means that the first antibody competes detectably with the second antibody (or other molecule) in a binding assay using either the recombinant KIR molecule or the surface-expressed KIR molecule. . For example, in one aspect, the percentage of inhibition for a particular antibody pair is about 20% or more, about 30% or more, about 30% or more, about 40% or more, regardless of which antibody was used as the first antibody. If greater than about 50%, the antibody competes. Alternatively, the antibodies compete if the percentage of inhibition for a particular antibody pair is averaged to at least about 29%, at least about 30%, at least about 40%, or at least about 50%. The percentage of inhibition of the binding of the second antibody to the KIR2D protein by the first antibody is 100 * (1- (detected binding of the second antibody) / (detected binding of the first antibody)) Can be calculated as The same applies to antibody fragments and antibody derivatives. Unless otherwise specified, antibodies that "competes" with 1-7F9, 1-4F1 or 1-7F9- or 1-4F1-like antibodies are human KIR2DL1, human KIR2DL2 / 3, or human KIR2DL1 and KIR2DL2 / It can compete with 1-7F9, 1-4F1 or 1-7F9- or 1-4F1-like antibodies for binding to both. For example, the antibody DF200 competes with 1-7F9 and 1-4F1 for binding to KIR2DL3.

  Optionally, the antibody that competes with 1-7F9 or 1-4F1 is not 1-7F9 or 1-4F1 respectively (ie, the invention binds to one or both of these KIRs in Provide antibodies other than 1-7F9 and 1-4F1, which are characterized, among others, by their ability to compete with 1-7F9 and / or 1-4F1)

  In another aspect, the antibody binds to both KIR2DL1 and KIR2DL2 / 3 human receptors and reduces or neutralizes or reverses inhibition of NK cell cytotoxicity mediated by these KIRs, Competes with 1-7F9 for binding to the KIR2DL2 / 3 human receptor, or to both the KIR2DL1 and KIR2DL2 / 3 human receptors. Optionally, the antibody is a chimeric antibody, a human antibody or a humanized antibody.

  In another aspect, the antibody binds to both KIR2DL1 and KIR2DL2 / 3 human receptors, reduces, neutralizes, or reverses the inhibition of NK cell cytotoxicity mediated by these KIRs, and KIR2DL1 Competes with EB6 for binding to the human receptor, or competes with GL183 and / or DF200 for binding to the KIR2DL2 / 3 human receptor, or competes with EB6 for binding to the KIR2DL1 human receptor and binds to the KIR2DL2 / 3 human receptor Competes with GL183 and / or DF200 for binding. In one embodiment, the antibody is not NKVSF1 (Pan2D), not A210, not A803g, and / or not DF200. The antibody is, for example, a murine, chimeric, human or humanized antibody.

  In another aspect, the antibody comprises a VH, VL, or both VH and VL regions that are at least substantially identical to the VH and / or VL regions of 1-7F9 or 1-4F1. The antibody may be of any subclass including IgG1, IgG2, IgG3, and IgG4. In a particular aspect, the antibody is a human IgG4 antibody. In another specific aspect, the antibody is a human IgG2 antibody. The antibody is a chimeric antibody, a human antibody or a humanized antibody.

  In another aspect, the antibody is a human antibody, competes with 1-7F9 or 1-4F1, and is at least partially the same or the same epitope on the KIR molecule as monoclonal antibody 1-7F9 or 1-4F1 Alternatively, it recognizes, binds, or has immunospecificity for an “epitope site”. Preferably, the KIR molecule is a human KIR2DL1 or KIR2DL2 / 3 receptor.

  In another aspect, the antibody binds to common determinants present on both KIR2DL1 and KIR2DL2 / 3 human receptors and neutralizes, reduces the inhibition of NK cell cytotoxicity mediated by these KIRs Or reverse. More specifically, the antibody can bind to the same, substantially the same, or the same epitope as monoclonal antibody 1-7F9 on KIR.

  In certain aspects, the antibody is a monoclonal antibody that exhibits one or more of the above properties.

  In another aspect, functional fragments and derivatives of the antibodies described herein can be prepared, which have substantially similar antigen binding, specificity and / or activity. , Including, but not limited to, Fab fragments, Fab′2 fragments, immunoadhesins, diabodies, camel antibodies, Janusins, minibodies, CDRs, and ScFv fragments.

  Unless otherwise specified, an antibody or a divalent fragment or derivative thereof is monospecific [ie, the “arms” of both the antibody, fragment, or derivative are the same antigen ( s)].

  In yet another aspect, antibody derivatives comprising the antibodies of the invention conjugated or covalently bound to toxins, radionuclides, detectable moieties (eg, fluorescent) or solid supports can be prepared.

  The invention also encompasses a pharmaceutical composition comprising an antibody, fragment thereof, or any derivative thereof as described above. Accordingly, the present invention also relates to the use of the antibodies disclosed herein in a method for producing a medicament. In a preferred embodiment, the medicament or pharmaceutical composition is for the treatment of cancer or other proliferative disorders, infections, or for use in transplantation.

  In one aspect, the present invention provides a composition [eg, a composition formulated for pharmaceutical administration, a kit for preparing such a composition, an assay kit or medium, a purification media, And the like, wherein the composition binds to at least two different human inhibitory KIR receptor gene products and is KIR-mediated for cytotoxicity by NK cells expressing at least one of the two different human inhibitory KIR receptors. Have the ability to neutralize inhibition of (wherein the antibody has been introduced into the liposomes). The liposome is, for example, a nucleic acid molecule for delivering a gene for gene therapy; a nucleic acid molecule for delivering an antisense RNA, RNAi or siRNA for suppressing a gene in an NK cell; or targeting an NK cell It may contain toxins or drugs to kill.

  Also described herein is a method for controlling human NK cell activity in vitro, ex vivo or in vivo, comprising an effective amount of an antibody of the invention, a fragment of such an antibody, of these A method comprising contacting human NK cells with any derivative or pharmaceutical composition comprising at least one of any of these. A preferred method comprises administering an effective amount of a pharmaceutical composition of the present invention, most preferably in a patient having cancer, an infectious disease or an immune disease, most preferably ex vivo or in vivo, human NK. It aims to increase the cytotoxic activity of cells. For example, administration can be accomplished by infusion of the antibody in an appropriate buffer via a vein into a patient with cancer or an infectious disease (eg, viral disease).

  The invention includes an antibody that binds at least two different human inhibitory KIR receptor gene products and a pharmaceutically acceptable carrier or excipient, wherein the antibody is one of the two different human inhibitory KIR receptors. In NK cells expressing at least one, it is possible to neutralize the inhibition of NK cell cytotoxicity mediated by KIR, wherein said antibody is NK cell in a patient or biological sample comprising NK cells. Also provided is a composition that is present in an amount effective to detectably enhance the cytotoxicity of Preferably, the antibody binds to a common determinant present on KIR2DL1 and KIR2DL2 / 3. The composition containing the antibody of the present invention may optionally contain a second therapeutic agent [related to the condition or disorder to which the antibody is administered (eg, cancer, transplantation, infectious disease, viral infection, etc. A drug that induces, promotes and / or enhances a therapeutic effect in a host associated with the condition). In one aspect, the second therapeutic agent that can be co-administered with the antibody of the present invention in such a combination composition is, for example, an immunomodulator, a hormonal agent, a chemotherapeutic agent, an anti-angiogenic agent (anti-angiogenic agent). agent), an apoptotic agent, a second antibody specific for a non-KIR antigen, optionally a second antibody that binds and inhibits and neutralizes or reduces signaling from an inhibitory KIR It can be selected from antibodies, anti-infectives, targeting inducers, or adjunct compounds. Advantageous immunomodulators include IL-1alpha, IL-1beta, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10 , IL-11, IL-12, IL-13, IL-15, IL-21, TGF-beta, GM-CSF, M-CSF, G-CSF, TNF-alpha, TNF-beta, LAF, TCGF, BCGF , TRF, BAF, BDG, MP, LIF, OSM, TMF, PDGF, IFN-alpha, IFN-beta, or IFN-gamma. Examples of the chemotherapeutic agents include alkylating agents, antimetabolites, cytotoxic antibiotics, adriamycin, dactinomycin, mitomacin, carminomycin, daunomycin, doxorubicin, tamoxifen, taxol, taxotere, vincristine, vinblastine, vinorelbine, Etoposide (VP-16), 5-fluorouracil (5FU), cytosine arabinoside, cyclophosphamide, thiotepa, methotrexate, camptothecin, actinomycin-D, mitomycin C, cisplatin (CDDP), aminopterin, combretastatin, others Vinca alkaloids and their derivatives or prodrugs are included. Examples of hormonal agents are leuprorelin, goserelin, triptorelin, buserelin, tamoxifen, toremifene, flutamide, nilutamide, cyproterone, bicalutamide anastrozole, exemestane, letrozole, fadrazole medroxy, chlormadinone, megestrol, other LHRH agonists , Other antiestrogens, other antiandrogens, other aromatase inhibitors and other progestagens. Preferably, said second antibody that binds to and inhibits an inhibitory KIR receptor is bound by said antibody that binds a common determinant present on at least two different human inhibitory KIR receptor gene products. An antibody or derivative or fragment of an antibody that binds to an epitope of an inhibitory KIR receptor that is different from the epitope of interest. In another aspect, the second antibody is directed to a target associated with a disease state (eg, cancer-associated antigen, virus-associated antigen, etc.) that is at least partially treatable by administration of an antibody of the invention. It may be.

  The present invention further provides a method for detectably enhancing NK cell activity comprising the step of administering the composition of the present invention to a patient in need of detectably enhancing NK cell activity. For patients in need of enhanced NK cell activity, such enhancement may promote, enhance and / or induce a therapeutic effect (or have a disease or disorder and be substantially similar to the patient). Any of those diagnosed with a disease or disorder that promotes, enhances, and / or induces such an effect in at least a significant proportion of patients with distinct characteristics (eg, as determined by clinical trials) Can be a patient. A patient in need of such treatment may suffer from, for example, cancer, another proliferative disease, an infectious disease or an immune disorder. Preferably, the method comprises an immunomodulator, a hormonal agent, a chemotherapeutic agent, an anti-angiogenic agent, an apoptotic agent, a second antibody specific for an antigen different from KIR, and optionally an inhibitory KIR receptor. A suitable additional therapeutic agent selected from a second antibody, an anti-infective agent, a target inducer or an adjunct compound that binds and inhibits and neutralizes or reduces signaling from an inhibitory KIR The additional therapeutic agent is administered to the patient in a single dosage form with the antibody or as a separate dosage form. The administration of the antibody (or antibody fragment / derivative) and the administration of the additional therapeutic agent both detectably induce, promote and / or enhance a therapeutic response in the patient, including enhanced NK cell activity. Enough. When administered separately, the antibody, fragment or derivative and the additional therapeutic agent are under conditions that provide the patient with a total detectable therapeutic utility (eg, with respect to timing, number of doses). Desirably administered.

  Further encompassed by the present invention are antibodies capable of specifically binding non-human primates, preferably monkey NK cells and / or KIR receptors. Also included are methods of assessing the toxicity, dosage and / or activity or efficacy of the antibodies of the invention that are candidate medicaments. In one aspect, the present invention provides administration of an antibody of the present invention to a non-human primate recipe endo animal having NK cells, wherein the drug is toxic or harmful to the animal, or preferably to a target tissue. It includes a method of determining the dose of an antibody that is toxic to an animal or target tissue by assessing any beneficial or adverse effect. In another aspect, the invention administers an antibody of the invention to a non-human primate recipe endo animal having NK cells, wherein the drug is toxic to the animal, or preferably to the target tissue, or A method of identifying antibodies that are toxic to animals or target tissues by assessing any harmful or adverse effects. In another aspect, the present invention comprises administering an antibody of the present invention to a non-human primate model of infection, disease or cancer, and identifying an antibody that reduces said infection, disease or cancer or symptoms thereof. To identify antibodies that are effective in treating infected diseases or tumors. In one embodiment, said antibody of the invention (a) cross-reacts with at least two inhibitory human KIR receptors present on the surface of human NK cells, and (b) non-human primate NK cells or KIRs An antibody that cross-reacts with a receptor.

Further encompassed by the present invention is a method for detecting the presence of NK cells having an inhibitory KIR on the cell surface in a biological sample or organism, comprising:
a) contacting said biological sample or organism with an antibody of the invention conjugated or covalently conjugated to a detectable moiety;
b) detecting the presence of the antibody in the biological sample or organism.

The present invention is a method for purifying NK cells having inhibitory KIR on the cell surface from a sample,
a) The antibody of the present invention conjugated or covalently bound to a solid support (eg, bead, matrix, etc.) under conditions that allow the NK cells having inhibitory KIR on the cell surface to bind to the antibody. Contacting the sample;
b) eluting the bound NK cells from the antibody conjugated or covalently bound to a solid support.

  The antibody NKVSF1 (Pan2D) was found to also bind to NK cells obtained from cynomolgus monkeys (see Example 10).

  Accordingly, the present invention provides antibodies that cross-react with at least two inhibitory human KIR receptors present on the surface of human NK cells and also bind to cynomolgus monkey-derived NK cells, and fragments and derivatives thereof. In one embodiment thereof, the antibody is not NKVSF1, is not A210, and / or is not A802g. The present invention is a method for examining the toxicity of antibodies cross-reacting with at least two inhibitory human KIR receptors present on the surface of human NK cells, and fragments and derivatives thereof, comprising examining said antibodies in cynomolgus monkeys A method is also provided.

  In a further aspect, the invention provides an antibody, antibody fragment or antibody or antibody fragment derivative comprising the light chain variable region or one or more light chain variable region CDRs of antibody 1-7F9 or 1-4F1. In yet another aspect, the invention provides an antibody, antibody fragment or antibody or antibody fragment comprising a sequence highly similar to all or substantially all of the light chain variable region sequence of 1-7F9 or 1-4F1 Derivatives of

  In a further aspect, the present invention provides an antibody, antibody fragment or antibody or antibody fragment derivative comprising the heavy chain variable region or one or more heavy chain variable region CDRs of antibody 1-7F9 or 1-4F1. In yet another aspect, the invention provides an antibody, antibody fragment or antibody or antibody fragment comprising a sequence highly similar to all or substantially all of the heavy chain variable region sequence of 1-7F9 or 1-4F1 Derivatives of

Antibodies The present invention binds to common determinants conserved among human inhibitory KIR receptors, preferably including determinants present on at least two different KIR2DL gene products but not on KIR2DS4. Novel antibodies and fragments or derivatives thereof are provided that cause potentiation of NK cells expressing at least one of these KIR receptors. The present invention produces such cross-reacting neutralizing antibodies that show unexpected results and open the way to new and effective therapies based on NK, particularly in human subjects, and modulate NK cell activity. It is disclosed for the first time that it can be used effectively.

  In the present invention, “common determinant” refers to a determinant or epitope shared by multiple gene products of the human inhibitory KIR receptor. Preferably, the common determinant is shared by at least two members of the KIR2DL receptor group. More preferably, the determinant is shared by at least KIR2DL1 and KIR2DL2 / 3. In addition to recognizing multiple gene products of the KIR2DL type, some antibodies of the invention are present on other inhibitory KIRs, such as gene products of the KIR3DL receptor group (eg, KIR3DL1 and / or KIR3DL2). Also recognizes determinants. A determinant or epitope can represent a peptide fragment or a conformational epitope shared by the members. In a more specific embodiment, the antibody of the present invention specifically binds to substantially the same epitope recognized by monoclonal antibody DF200. This determinant is present on both KIR2DL1 and KIR2DL2 / 3. In another preferred embodiment, the antibodies of the invention specifically bind to substantially the same epitope recognized by human mAb 1-7F9 or to an epitope recognized by human mAb 1-4F1. The epitope is present on KIR2DL1, -2 and -3 but not on KIR2DS4.

  As used herein, the term “antibody” refers to polyclonal and monoclonal antibodies, as well as fragments and derivatives of the polyclonal and monoclonal antibodies, unless otherwise specified or clearly stated to the contrary. . Depending on the type of constant domain in the heavy chain, full-length antibodies are typically assigned to one of five major classes: IgA, IgD, IgE, IgG, and IgM. Some of these are further divided into subclasses or isotypes, such as IgG1, IgG2, IgG3, IgG4, and the like. The heavy-chain constant domains that correspond to the different classes of immunoglobulins are called “α”, “δ”, “ε”, “γ”, and “μ”, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known. IgG and / or IgM are the preferred class of antibodies used in the present invention because they are the most common antibodies in physiological situations and are most easily made in laboratory conditions. Typically, “antibody” in the present invention means a monoclonal antibody, more preferably a “human” monoclonal antibody.

  One aspect of the invention provides a method for therapeutically blocking in vivo interaction between an inhibitory KIR and its corresponding HLA ligand, which involves the activation of endogenous NK cells. To do. In such a setting, it would be advantageous to avoid depletion of NK cells. This is because, when depleted, the NK cells will not be able to exert their therapeutically useful effects. Accordingly, those that exhibit some binding to Fc receptors, such as antibody isotypes, IgG4 and IgG2, and do not activate the complement system are typically preferred. The isotype of 1-7F9 is IgG4. In addition, IgG2 antibodies are generally considered non-depleting and can be more stable molecules than IgG4 antibodies, which confer a long half-life in vivo. . 1-4F1 is an IgG2 isotype.

Antibody Production The antibodies of the present invention can be made by various techniques known in the art. Typically, an antibody of the invention is made by immunizing a non-human animal, preferably a mouse, with an immunogen comprising an inhibitory KIR polypeptide, preferably a KIR2DL polypeptide, more preferably a human KIR2DL polypeptide. Is done. Said inhibitory KIR polypeptide is a full-length sequence of a human inhibitory KIR polypeptide, or a fragment or derivative thereof, typically an immunogenic fragment (ie, on the surface of a cell expressing an inhibitory KIR receptor). A portion of a polypeptide comprising an epitope exposed to Such fragments typically contain at least about 7 contiguous amino acids of the mature polypeptide sequence, more preferably at least about 10 contiguous amino acids of the mature polypeptide sequence. Fragments are typically substantially derived from the extracellular domain of the receptor. Further preferred is a human KIR2DL polypeptide that contains at least one, more preferably both of the extracellular Ig domains of the full-length KIRDL polypeptide and can mimic at least one conformational epitope present in the KIR2DL receptor. is there. In other embodiments, the polypeptide comprises at least about 8 contiguous amino acids of the extracellular Ig domain at amino acid positions 1-224 of the KIR2DL1 polypeptide [Wagtmann, incorporated herein by reference in its entirety. (Wagtmann et al., Immunity 1995; 2: 439-449) or found at the World Wide Web (www) address ncbi.nlm.nih.gov/prow/guide/1326018082.htm Amino acid numbering according to site].

  In one embodiment, the immunogen comprises a wild type KIR2DL polypeptide in a lipid membrane, typically on the surface of a cell. In a specific embodiment, said immunogen comprises intact NK cells, in particular intact human NK cells. In another embodiment, the cells are lysed or processed so that they are not intact. The immunogen can be, for example, a non-human mammal (eg, mouse, rabbit, goat, horse, dog, sheep, guinea pig, rat, hamster, etc.) in a buffer (optionally an adjuvant such as Freund's adjuvant). ) Can be suspended or dissolved prior to administration.

  The step of immunizing a non-human mammal with an antigen can be performed in any manner known in the art to stimulate antibody production in mice (eg, E. Harlow and D. Lane , Antibodies: A Laboratory Manual., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1988)). The immunogen is then suspended or dissolved in a buffer, optionally with an adjuvant, such as complete Freund's adjuvant. Methods for determining the amount of immunogen, type of buffer and amount of adjuvant are well known to those skilled in the art and are not intended to limit the invention in any way. These parameters can be different for different immunogens, but are easily elucidated.

  Similarly, the principles for selecting the location and frequency of immunization sufficient to stimulate antibody production are also well known in the art. In an exemplary immunization protocol, on day 1, non-human animals are injected intraperitoneally with the antigen, and about 1 week later, the antigen is injected intraperitoneally again. Thereafter, if necessary, recall injections of the antigen are performed around day 20 together with an adjuvant such as incomplete Freund's adjuvant. Recall injections are given intravenously and can be repeated for several consecutive days. Thereafter, a booster injection is usually given on day 40, either intravenously or intraperitoneally, without adjuvant. This protocol produces antigen-specific antibody-producing B cells after about 40 days. Other protocols can be used as long as they result in the production of B cells that express antibodies induced against the antigen used for immunization.

  For the preparation of polyclonal antibodies, serum is obtained from the immunized non-human animal and the antibodies present therein are isolated by well known techniques. To obtain an antibody that reacts with an inhibitory KIR receptor, serum can also be affinity purified using any of the above immunogens linked to a solid support.

  In another embodiment, lymphocytes are isolated from non-immunized non-human mammals, grown in vitro, and then exposed to the immunogen in cell culture. The lymphocytes are then collected and the fusion process described below is performed.

  In the case of monoclonal antibodies, the next step is to isolate spleen cells from the immunized non-human mammal and then fuse these spleen cells with immortalized cells to form a hybridoma that produces the antibody. . Isolation of spleen cells from non-human mammals is well known in the art and typically involves removing the spleen from an anesthetized non-human mammal, cutting the spleen into small pieces, and splenic capsules. ) Through the nylon mesh of the cell strainer and squeeze the splenocytes into a suitable buffer to obtain a single cell suspension. Cells are washed, centrifuged and resuspended in a buffer that lyses all red blood cells. This solution is centrifuged again and finally the lymphocytes remaining in the pellet are resuspended in fresh buffer.

  Once isolated and present in a single cell suspension, lymphocytes can be fused to an immortal cell line. This is typically a murine myeloma cell line, but many other immortal cell lines useful for the production of hybridomas are also known in the art. Preferred mouse myeloma lines include MOPC-21 and MPC-11 mouse tumors (available from Salk Institute Cell Distribution Center, San Diego, Calif. USA), or X63, Ag8653 and SP-2 cell lines (American Type Culture Collection). , Available from Rockville, Maryland USA), but is not limited to these. Cell fusion is performed using polyethylene glycol or the like. The resulting hybridoma is then grown in a selective medium containing one or more substances that inhibit the growth or survival of unfused parental myeloma cells. For example, if the parent myeloma cells lack the enzyme hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), the medium for hybridomas typically contains hypoxanthine, amino acids, which are substances that suppress the growth of HGPRT-deficient cells. Will contain pterin and thymidine (HAT medium).

  Hybridomas are typically grown on macrophage feeder cells. Macrophages are preferably obtained from littermates of non-human mammals used to isolate splenocytes and are typically stimulated with incomplete Freund's adjuvant or the like a few days before seeding the hybridoma . Fusion methods are described in Goding (Monoclonal Antibodies: Principles and Practice, "pp. 59-103 (Academic Press, 1986), the disclosure of which is incorporated herein by reference.

  The cells are grown in selective media for a time sufficient for colony formation and antibody production. This is usually between about 7 and about 14 days. The hybridoma colonies are then assayed for the production of antibodies that cross-react with multiple inhibitory KIR receptor gene products. The assay is a typical colorimetric ELISA type assay, but may be performed in several other types of assays, including immunoprecipitation and radioimmunoassay, or FACS, Biacore, scintillation proximity Assays (SPA) or other types of assays known in the art are included. To determine if one or more different hybridoma cell colonies are present, the wells containing antibodies of the desired specificity are examined. If more than one colony is present, the cells can be recloned and only a single cell can be grown to ensure that a colony producing the desired antibody is produced. In order to ensure that only one monoclonal antibody is detected and produced, typically positive cells with a single apparent colony are recloned and reassayed.

In preferred embodiments, non-human animals used to generate antibodies according to the applicable methods of the invention are rodents (eg, mice, rats, etc.), cows, pigs, horses, rabbits, goats. , Mammals such as sheep. In addition, non-human mammals can be genetically modified or engineered to produce “human” antibodies, such as Xenomous ^™ (Abgenix) or HuMAb-Mouse ^™ (Medarex), as described below. can do.

  The antibody also produces the antibody in a recoverable form through and from the production of a chordate (eg, mammal or bird) or plant that is transgenic for the heavy and light chain sequences of the desired immunoglobulin. [For example, for the production of antibodies and antibody-like proteins in plants, Ma et al., Nature Rev. Genetics 4: 794-805 (2003); Nolke et al., Expert Opin Biol Ther. 2003 Oct; 3 (7): 1153-62; Schillberg et al., Cell Mol Life Sci. 2003 Mar; 60 (3): 433-45; Tekoah et al., Arch Biochem Biophys. 2004 Jun 15 426 (2): 266-78; Fischer et al., Eur. J. of Biochem., 262 (3): 810 (1999); and US Patent Application 20030084482]. In connection with transgenic production in mammals, antibodies and other proteins can be produced in and recovered from milk from goats, cows, or other mammals. See U.S. Pat. Nos. 5,827,690, 5,756,687, 5,750,172, and 5,741,957. The antibody may also be produced in and recovered from avian eggs. See, for example, Tini et al. (Tini et al., Comp Biochem Physiol A Mol Integr Physiol. 2002 Mar; 131 (3): 569-74) and US Pat. No. 4,550,019.

  Antibodies can also be generated by selecting a combinatorial library of immunoglobulins, for example as disclosed in Ward et al. (Ward et al., Nature, 341 (1989) p. 544).

  According to another embodiment, the present invention produces antibodies capable of binding determinants present on at least two different human inhibitory KIR receptor gene products to neutralize the inhibitory activity of the receptor. A hybridoma derived from a B cell obtained from a non-human host is provided. More preferably, the hybridoma of this aspect of the invention is not a hybridoma that produces monoclonal antibodies NKVSF1, A210, and / or A802g. The hybridoma according to this aspect of the present invention can be produced as described above by fusing spleen cells obtained from an immunized mammal other than a human with an immortal cell line. Hybridomas produced by this fusion can be screened for the presence of such cross-reacting antibodies, as described elsewhere herein. Preferably, the hybridoma recognizes determinants present on at least two different KIR2DL gene products and produces antibodies that cause enhancement of NK cells expressing at least one of these KIR receptors. More preferably, said hybridoma produces an antibody that binds to substantially the same epitope or determinant as 1-7F9 and enhances NK cell activity or binds to substantially the same epitope as 1-4F1. Most preferably, the hybridoma is a hybridoma 1-7F9 that produces monoclonal antibody 1-7F9 or a hybridoma 1-4F1 that produces monoclonal antibody 1-4F1.

  Hybridomas that have been confirmed to produce the monoclonal antibodies of the invention can be grown in larger quantities in a suitable medium such as DMEM or RPMI-1640. Alternatively, the hybridoma cells can be grown in vivo as ascites cancer in an animal.

  After sufficient growth until the desired monoclonal antibody is produced, the growth medium (or ascites fluid) containing the monoclonal antibody can be separated from the cells and the monoclonal antibody is purified. Typically, purification is performed by chromatography using anti-mouse Ig linked to a solid support such as Protein A or Protein G-Sepharose, or agarose or Sepharose beads (all, eg, “Antibody Purification Handbook, Amersham Biosciences, publication No. 18-1037-46, Edition AC, the disclosure of which is incorporated herein by reference), or other known such as gel electrophoresis or dialysis Achieved through technology. The bound antibody is typically eluted from the Protein A / Protein G column by using a low pH buffer (glycine or acetate buffer at pH 3.0 or lower), and the antibody-containing fraction immediately in the middle. To sum up. If necessary, these fractions can be pooled, dialyzed and concentrated.

In one aspect of the human antibody, the present invention provides a human anti-KIR antibody. “Human” antibodies can be distinguished from “humanized” antibodies (these are described separately below). Such “human” antibodies may comprise amino acid residues that are not encoded by human germline immunoglobulin sequences, eg, in CDRs such as CDR3 (eg, random or site-directed mutagenesis in vitro or in the body. Mutations introduced by carrying out cell mutations in vivo). However, the term “human antibody” as used herein is a humanized antibody or a human / mouse chimeric antibody and is derived from the germline of another mammalian species (eg, mouse). It is contemplated that CDR sequences do not include the antibody grafted into human framework sequences.

Transgenic animals carrying the human Ig gene can be developed and upon immunization produce a complete repertoire of human antibodies in the absence of mouse immunoglobulin production. Such human Ig-transgenic mice can be used to produce human antibodies. Such human antibodies may be used in human Ig-transgenic animals (eg, mice, rats, sheep, pigs, goats, cows, horses, etc.) that carry the human immunoglobulin locus and the native immunoglobulin gene deletion. can be produced ^{[e.g., XenoMouse TM (Abgenix- Fremont, CA} , USA) in] [e.g., Green et al Nature Genetics 7: .. 13-21 (1994); Mendez et al Nature Genetics 15: 146- 156 (1997); Green and Jakobovits J. Exp. Med. 188: 483-495 (1998); European Patent No. EP0463151B1; International Patent Application Numbers WO94 / 02602, WO96 / 34096; / 53049, and WO00 / 037504; and U.S. Pat. Transgenic animals [for example, HuMab-mouse ^TM (Medarex-Princeton , NJ, USA)] (e.g., EP 0546073, EP0546073; U.S. Pat. International Patent Application Nos. WO 92/03918, WO 92/22645, WO 92/22647, WO 92/22670, WO 93/12227, WO 94/00569, WO 94/25585, WO 96/14436, WO 97 / 13852, and WO 98/24884). Spleen cells from such transgenic mice can be used to produce hybridomas that secrete human monoclonal antibodies by well-known techniques described herein. Similar techniques and principles are described, for example, by Jakobovits et al. And Bruggemann et al [Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90: 2551-255 (1993); Jakobovits et al., Nature, 362: 255- 258 (1993); and Bruggemann et al., Year in Immuno., 7:33 (1993)].

  In addition, human antibodies or antibodies from other species can be obtained through non-limiting display-type techniques, including phage display, retroviral display, ribosome display, and other related techniques, using methods known in the art. Such techniques are also well known, and the resulting molecules can be subjected to additional maturation methods such as affinity maturation [see, eg, (Hoogenboom et al. , J. Mol. Biol. 227: 381 (1991) (phage display); Vaughan, et al., Nature Biotech 14: 309 (1996) (phage display); Hanes and Plucthau PNAS USA 94: 4937-4942 (1997) (Ribosome display), Parmley and Smith Gene 73: 305-318 (1988) (phage display), Marks et al., J. Mol. Biol., 222: 581 (1991), Scott TIBS 17: 241-245 (1992) ), Cwirla et al. PNAS USA 87: 6378-6382 (1990), Russel et al. Nucl. Acids Research 21: 1081-1085 (1993), Hoganboom et al. Immunol. Reviews 130: 43-68 (1992), Chiswell and McCafferty TIBTECH 10:80 -84 (1992), and U.S. Patent No. 5,733,743.] If display technology is utilized to produce non-human antibodies, such antibodies can be humanized (see, eg, herein). As described elsewhere).

  Thus, as described herein, anti-KIR mAbs are promising agents for the treatment of cancer and viral infections and other diseases and disorders. Anti-KIR mAbs are humanized mouse mAbs or by fusion of splenocytes from human Ig-transgenic mice (XenoMouse, or HuMab mice), by phage display, or immortalization of human Ab-producing B cells Or by various approaches such as other methods. In any case, the antibody can be produced in a cell line and purified in an appropriate amount for formulation, packaging, and injection into a patient in need thereof.

Recombinant production Anti-KIR antibodies may also be produced in unicellular organisms such as yeast; or bacterial cell cultures (eg E. coli); or eukaryotic cell cultures (eg mammalian cell cultures). Can be prepared by expressing the recombinant form using standard techniques.

  Thus, according to another aspect, the DNA encoding the heavy and light chains of an anti-KIR antibody that binds, cross-reacts and neutralizes determinants present on at least two different human inhibitory KIRs. Isolated from the hybridomas of the invention and placed in a suitable expression vector for transfection into a suitable host. The host is then used to recombinantly produce antibodies, or variants thereof, such as humanized forms of monoclonal antibodies, active fragments of antibodies, or chimeric antibodies that contain an antigen recognition portion of an antibody. Preferably, the DNA used in this embodiment recognizes determinants present on at least two different KIR2DL gene products but not on KIR2DS3 or -4, and at least one of these KIR receptors is It encodes an antibody that causes enhancement of expressed NK cells. More preferably, the DNA encodes an antibody that binds to substantially the same epitope or determinant as 1-7F9 and enhances NK cell activity. Most preferably, this DNA encodes monoclonal antibody 1-7F9.

  The DNA encoding the monoclonal antibody of the present invention uses oligonucleotide probes that can specifically bind to the genes encoding the heavy and light chains of mouse or human antibodies using conventional procedures. Easily) and sequenced. Once the DNA is isolated, the DNA can be placed in an expression vector, and then the expression vector is transformed into a host cell (an E. coli cell, monkey COS cell that does not produce immunoglobulin protein in an untransfected state). , In Chinese hamster ovary (CHO) cells or myeloma cells) to obtain monoclonal antibody synthesis in recombinant host cells. Recombinant expression in bacteria of DNA encoding a fragment of an antibody is well known in the art [see, eg, Skerra et al., Curr. Opinion in Immunol., 5, pp. 256 (1993); and Pluckthun, Immunol. Revs. 130, pp. 151 (1992)].

  Furthermore, recombinant production of antibodies from known variable heavy (VH) and variable light (VL) chains and human constant regions has been described, for example, by the following authors [Ruker et al. (Annals of the New York Academy of Sciences. 1991; 646: 212-219), he reports the expression of human monoclonal anti-HIV-1 antibodies in CHO cells; Bianchi et al. (Biotechnology and Bioengineering. 2003; 84: 439 -444), he describes that high-level expression of full-length antibodies was performed using trans-complementing expression vectors; No Soo Kim et al. (Biotechnol. Prog. 2001). 17: 69-75), he describes important determinants in the occurrence of clonal variation in humanized antibody expression in CHO cells during gene amplification mediated by dihydrofolate reductase; King et al. (Biochemical Journal. 1992; 281: 317-323), he is a mouse-human chimeric antibody and chimeric Fa reports expression, purification, and characterization of b'fragments; WO 2003064606 comprises human heavy and human light chain variable regions, both of which are FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4 sequences And WO2003040170 describes chimeric or human monoclonal antibodies and antigen-binding portions that specifically bind to and activate human CD40].

The entire cDNA sequence encoding the constant region of human IgG can be found in the following GenBank entries accessed on January 6, 2005, each of which is incorporated by reference in its entirety:
Human IgG1 constant heavy chain region: GenBank accession #: J00228
Human IgG2 constant heavy chain region: GenBank accession #: J00230
Human IgG3 constant heavy chain region: GenBank Accession #: X04646
Human IgG4 constant heavy chain region: GenBank accession #: K01316
Human kappa light chain constant region: GenBank accession #: J00241
In an exemplary embodiment, the following protocol can be applied to generate recombinant mAb production from VH and VL sequences of 1-7F9 or 1-4F1. Steps 1-3 describe the retrieval of VH and VL regions from hybridomas or other cells producing 1-7F9 or 1-4F1. In contrast, cDNAs encoding 1-7F9 or 1-4F1 VH and VL sequences (or variants or derivatives thereof) can be obtained from the sequence information provided in FIG. 14 or 15. Can be prepared using established techniques for synthesizing cDNA fragments. Alternatively, 1-7F9 or 1-4F1 VH and VL fragments, or mutants or derivatives thereof, contain several scientific regions that contain the constant region of the desired Ig subclass in order to express full-length antibodies. And may be cloned into any one of the expression vectors described in the literature or commercially available expression vectors. Additionally, 1-7F9 or 1-4F1 VH and VL fragments, or mutants or derivatives thereof, encode truncated constant regions to express antibody fragments (eg, Fab fragments). Can be cloned into the vector. An example of a commercially available vector is pASK84, commercially available from ATCC (American Type Culture Collection, catalog number 87094).

(1) Isolation of total RNA from hybridoma cells:
4 × 10 ⁶ hybridoma cells (eg, 1-7F9 or 1-4F1) secreting antibodies against human KIR are used for isolation of total RNA. This procedure is carried out according to the manufacturer's instructions using the RNeasy Mini Kit from Qiagen and is outlined below:
The cells are pelleted by centrifugation at 1000 rpm for 5 min and disrupted by the addition of 350 μl RLT buffer containing 10 μl / ml β-mercaptoethanol. Transfer the lysate to a QIAshredder column from Qiagen and centrifuge at maximum speed for 2 min. Mix the flow-through with an equal volume of 70% ethanol. For each RNeasy spin column (Qiagen), apply up to 700 μl of sample, centrifuge at 14000 rpm, and discard the flow-through. Apply 700 μl of RW1 buffer to each column centrifuged at 14000 rpm for 15 s to wash the column. The column is washed twice with 500 μl RPE buffer and centrifuged for 15 s at 14000 rpm. To dry the column, it is additionally centrifuged at 14000 rpm for 2 min. The column is transferred to a new collection tube. RNA is eluted with 50 μl nuclease-free water and centrifuged for 1 min at 14000 rpm. RNA concentration was measured by absorbance at OD = 260 nm. RNA is stored at -80 ° C until needed.

(2) cDNA synthesis:
1 μg of RNA was used for first strand cDNA synthesis using SMART RACE cDNA Amplification Kit (Clontech). For the preparation of 5'-RACE-Ready cDNA, a reaction mixture was prepared and RNA isolated as described above, reverse-primer 5'-CDS primer back, and SMART II A Contains oligos. This mixture was then incubated at 72 ° C. for about 2 min, then cooled on ice for about 2 min, and 1 × First-Strand buffer, DTT (20 mM), dNTP (10 mM) and PowerScript reverse transcriptase were added. The reaction mixture is incubated at 42 ° C for 1.5 hours. Then, tricine-EDTA buffer was added and incubated at 72 ° C for 7 min. At this point, the sample can be stored at -20 ° C.

(3) PCR amplification and cloning of human variable light chain (VL) and human variable heavy chain (VH):
A PCR (Polymerase Chain Reaction) reaction mixture containing 1xAdvantage HF 2 PCR buffer, dNTP (10 mM) and 1xAdvantage HF 2 polymerase mix was used for variable regions of both VL and VH from cDNA generated as described above. Achieved for separate amplification.

For VL amplification, the following primers are used:
UPM (Universal Primer Mixture):
5'-CTAATACGACTCACTATAGGGCAAGCAGTGGTATCAACGCAGAGT-3 '(SEQ ID NO: 26)
5'-CTAATACGACTCACTATAGGG-3 '(SEQ ID NO: 27)
VK RACE2:
5'-GCAGGCACACAACAGAGGCAGTTCCAGATTTC-3 '(SEQ ID NO: 28)
For VH amplification, the following primers are used:
UPM (Universal Primer Mixture):
5'-CTAATACGACTCACTATAGGGCAAGCAGTGGTATCAACGCAGAGT-3 '(SEQ ID NO: 29)
5'-CTAATACGACTCACTATAGGG-3 '(SEQ ID NO: 30)
AB90RACE:
5'-GTGCCAGGGGGAAGACCGATGGG-3 '(SEQ ID NO: 31)
PCR was performed for 3 rounds. Round 1: PCR was performed for 5 cycles at 94 ° C for 5 s and 72 ° C for 3 min. Round 2: PCR was performed for 5 cycles at 94 ° C for 5 s, 70 ° C for 10 s, and 72 ° C for 1 min. Round 3: PCR was performed for 28 cycles at 94 ° C for 5 s, 68 ° C for 10 s, and 72 ° C for 1 min.

  The PCR product was analyzed by electrophoresis on a 1% agarose gel and DNA was purified from the gel using the QIAEX11 agarose gel extraction kit (from Qiagen).

  The purified PCR product was introduced into PCR4-TOPO using a TOPO TA cloning kit (from Invitrogen) and used for transformation of TOP10 competent cells.

The appropriate amount of colonies was analyzed by colony PCR using Taq polymerase, 1xTaq polymerase buffer, dNTP (10 mM) and the following primers and PCR program:
M13 forward primer: 5'-GTAAAACGACGGCCAG-3 '(SEQ ID NO: 32)
M13 reverse primer: 5'-CAGGAAACAGCTATGAC-3 '(SEQ ID NO: 33)
PCR program:
25 cycles were performed at 94 ° C for 30 s, 55 ° C for 30 s, and 72 ° C for 1 min.

  Plasmid DNA from clones carrying VL and VH inserts, respectively, was extracted and sequenced using the primers M13 forward and M13 reverse described above. In the case of human anti-KIR mAb 1-7F9, the sequences encoding the heavy and light chain variable regions are shown in FIG.

(4) Subcloning of the antibody gene into a mammalian expression vector Based on the sequence data on the cDNAs encoding the variable region of the heavy chain and light chain of the mAb, the primers were variable light chain (VL) and variable heavy chain ( VH) designed for gene amplification. The variable region is formatted with PCR and contains a Kozak sequence, a leader sequence and a unique restriction enzyme site. For the VL, this was accomplished by designing 5 ′ PCR primers to introduce a HindIII site, a Kozak sequence, and be homologous to the 5 ′ end of the leader sequence of the variable light chain region. The 3 ′ primer is homologous to the 3 ′ end of the variable region and a BsiWI site was introduced at the 3 ′ boundary of the variable region. The VH region was created in a similar manner. However, NotI and NheI sites were introduced in place of HindIII and BsiWI at the 5 ′ and 3 ′ ends, respectively.

  The amplified genes were each cloned using standard techniques into eukaryotic expression vectors containing light and heavy chain constant regions. The DNA fragment of VL is digested with HindIII and BsiWI and ligated into a eukaryotic expression vector containing the beta-lactamase gene encoding ampicillin resistance and the E. coli replication origin (pUC); The resulting plasmid was named VLCL. The VH DNA fragment was digested with NotI and NheI and introduced into the VLCL vector generated by the introduction of the VL fragment. The resulting plasmid contains a functional expression cassette that encodes both the heavy and light chains of the antibody on the same plasmid. The ligated plasmid was used to transform E. coli. Plasmid DNA was prepared from these ampicillin resistant bacterial populations and used for transfection into Chinese hamster ovary cells or other mammalian cell lines. Transfection and cell culture were performed, for example, by standard methods described in Sambrook et al. (“Molecular Cloning”, Sambrook et al.). Stable expression of a desired antibody, such as a 1-7F9 or 1-4F1 human anti-KIR mAb or a mAb containing the VH and VL regions of 1-7F9 or 1-4F1, or another human anti-KIR mAb A secreted transfected cell line resulted.

  Antibody variants can be easily generated. For example, an antibody of an isotype that has exactly the same specificity as 1-7F9 or 1-4F1, respectively, but different from IgG4 or IgG2, is responsible for the cDNA encoding VL and VH of 1-7F9 or 1-4F1, It can be obtained by subcloning into a plasmid containing a kappa light chain constant region and a heavy chain constant region selected from the constant heavy chain regions of IgG1, IgG2, IgG3 or IgG4. Therefore, the produced antibody may have any isotype. Moreover, the said antibody can switch an isotype using the normal technique in the said technique. Such techniques include direct recombination techniques (see, eg, US Pat. No. 4,816,397), cell-cell fusion techniques (see, eg, US Pat. No. 5,916,771), and other suitable techniques known in the art. Includes the use of technology. Thus, the effector functions of the antibodies provided by the present invention may be used for a variety of uses, including therapeutic ones with respect to the parent antibody isotype, eg, IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE, or It may be “changed” by isotype switching to an IgM antibody.

  In a further aspect, the present invention is obtained from a mammalian host immunized with an antigen comprising an epitope present on an inhibitory KIR polypeptide (typically a non-human mammalian host), and (b) immortalized. A hybridoma comprising (a) a B cell fused to a cultured cell (eg, a myeloma cell), said hybridoma binding at least two different human inhibitory KIR receptors and said at least two different humans In a population of NK cells that express an inhibitory KIR receptor, a monoclonal antibody is produced that can at least substantially neutralize KIR-mediated inhibition of NK cell cytotoxicity. In one embodiment, the mammalian host is a transgenic animal capable of producing human antibodies. Optionally, the hybridoma does not produce the monoclonal antibody NKVSF1, does not produce A210, and / or does not produce A802g. In various embodiments, the antibody binds to common determinants present on KIR2DL1 and KIR2DL2 / 3 receptors or on both KIR2DL1 and KIR2DL2 / 3. The hybridoma binds an HLA-C allele molecule having a Lys residue at position 80 to a human KIR2DL1 receptor and an HLA-C allele molecule having an Asn residue at position 80 to a human KIR2DL2 / 3 receptor. Antibodies can be produced that inhibit binding. For example, the hybridoma may produce an antibody that binds to substantially the same epitope as monoclonal antibody 1-7F9 or 1-4F1 on either KIR2DL1 or KIR2DL2 / 3 or both KIR2DL1 and KIR2DL2 / 3.

The present invention also provides a method of making an antibody that cross-reacts with a plurality of KIR2DL gene products to reduce or neutralize the inhibitory activity of such KIR, comprising the following steps:
(A) immunizing a non-human mammal with an immunogen comprising a KIR2DL polypeptide;
(B) preparing an antibody that binds to the KIR2DL polypeptide from the immunized mammal;
(C) selecting an antibody of (b) that cross-reacts with at least two different KIR2DL gene products;
(D) A step of selecting the antibody of (c) that enhances NK cells. In one embodiment, the non-human mammal is a transgenic animal engineered to express a human antibody repertoire [eg, a non-human having a deletion of a human immunoglobulin locus and a unique immunoglobulin gene. Mammals, Xenomouse ^™ (Abgenix-Fremont, CA, USA), etc., or non-human mammals having a microlocus of genes encoding human Ig, HuMab-Mouse ^™ (Medicarex-Princeton, NJ, USA) )Such〕. If desired, steps a and b may be replaced by alternative methods of obtaining anti-KIR mAbs, including but not limited to the use of phage display or viral transduction of human B cells, Or other methods known in the art. Optionally, the method further comprises selecting an antibody that binds to a KIR, NK cell or KIR polypeptide in a primate, preferably a cynomolgus monkey. If necessary, the present invention further relates to a method for evaluating an anti-KIR antibody, wherein the antibody produced according to the above method is administered to a primate, preferably a cynomolgus monkey, Includes methods observed for the presence or absence of toxicity indicators.

The present invention is a method of making an antibody that binds at least two different human inhibitory KIR receptor gene products, wherein said antibody expresses said at least two different human inhibitory KIR receptor gene products. Having the ability to neutralize (or enhance NK cytotoxicity) the KIR-mediated inhibition of cytotoxicity by a population of
a) immunizing a non-human mammal with an immunogen comprising an inhibitory KIR polypeptide;
b) preparing an antibody that binds the KIR polypeptide from the immunized animal;
c) selecting an antibody of (b) that cross-reacts with at least two different human inhibitory KIR receptor gene products;
Selecting the antibody of (c) capable of neutralizing KIR-mediated inhibition of NK cell cytotoxicity against a population of NK cells expressing said at least two different human inhibitory KIR receptor gene products Wherein the order of steps (c) and (d) is reversed as necessary, and any number of the steps are repeated one or more times as needed. The inhibitory KIR polypeptide used for immunization may in one aspect be an antibody selected in step (c) that cross-reacts with one or more KIR2DL polypeptides and at least KIR2DL1 and KIR2DL2 / 3. Preferably, the antibody recognizes common determinants present on at least two different KIR receptor gene products, and most preferably, the KIR is KIR2DL1 and KIR2DL2 / 3. In one embodiment, step (c) comprises selecting an antibody that reacts with at least one KIR2DL and one KIR3DL receptor gene product. For example, the selected antibody could react with KIR2DL1 and KIR2DL2 / 3 as well as KIR3DL1 and / or KIR3DL2. Optionally, the method further comprises selecting an antibody that binds a primate, preferably cynomolgus monkey NK cell or KIR polypeptide. Optionally, the present invention further provides a method for evaluating an antibody, wherein an antibody produced according to the above method is administered to a primate, preferably a cynomolgus monkey, and preferably said monkey is toxic for said antibody. Includes methods that are observed for the presence or absence of an indicator.

  Optionally, in the above method, the antibody selected in step c) or d) is not NKVSF1, is not A210, and / or is not A802g. Preferably, the antibody prepared in step (b) of the above method is a monoclonal antibody. Preferably, the antibody selected in step (c) of the above method has one or more HLA-C allotypes having a Lys residue at position 80 binding to the human KIR2DL1 receptor and an Asn residue at position 80 Inhibits binding of one or more HLA-C allotypes to the human KIR2DL2 / 3 receptor. Preferably, the antibody selected in step (d) of the above method results in an enhancement in NK cytotoxicity or neutralization of NK cytotoxic KIR-mediated inhibition. Preferably, the antibody binds to substantially the same epitope on KIR2DL1 and / or KIR2DL2 / 3 as monoclonal antibody 1-7F9. Where appropriate, the method may include generating a fragment of a selected monoclonal antibody in addition to or in place of the step and conjugating it to (eg, a radionuclide, cytotoxic agent, reporter molecule, etc.). The addition of creating a derivative of the selected monoclonal antibody or generating a derivative of the antibody fragment generated from the sequence corresponding to the sequence of such a monoclonal antibody or comprising a sequence corresponding to the sequence of such a monoclonal antibody These steps are included. In another aspect, the variant of the antibody increases the stability of the antibody, which alters the amino acid sequence of the variant obtained from the antibody obtained by the method described above (eg, alters the binding properties of the antibody with respect to the administered host). An antibody comprising a known gene, comprising a sequence modified to reduce or enhance the purification of the antibody, enhance the detection of the antibody, and / or alter the immunological properties of the antibody. Can be produced using standard operating procedures.

Furthermore, the present invention is a method of making an antibody that binds at least two different human inhibitory KIR receptor gene products, said antibody expressing at least one different human inhibitory KIR receptor gene product. The inhibition of NK cell cytotoxicity mediated by KIR in a population of cells can be neutralized (or enhanced by NK cytotoxicity), the method comprising the following steps:
(A) selecting from a library or repertoire monoclonal antibodies or antibody fragments that cross-react with at least two different human inhibitory KIR2DL receptor gene products;
(B) the antibody of (a) capable of neutralizing KIR-mediated inhibition of NK cell cytotoxicity in a population of NK cells expressing said at least two different human inhibitory KIR2DL receptor gene products; And a step of selecting. Preferably, the antibody binds to a common determinant present on KIR2DL1 and KIR2DL2 / 3. Optionally, the antibody selected in step (b) is not NKVSF1. Preferably, the antibody selected in step (b) is such that the HLA-C allele molecule having a Lys residue at position 80 binds to the human KIR2DL1 receptor and the HLA-C allele has an Asn residue at position 80. Inhibits the binding of the molecule to the human KIR2DL2 / 3 receptor. Preferably, said antibody selected in step (b) produces an increase in NK cytotoxicity. Preferably, the antibody binds to substantially the same epitope on KIR2DL1 and / or KIR2DL2 / 3 as monoclonal antibody 1-7F9. Alternatively, the antibody binds to substantially the same epitope as monoclonal antibody 1-4F1. Optionally, the method further comprises producing a fragment of the selected monoclonal antibody, producing a derivative of the selected monoclonal antibody, or producing a derivative of the selected monoclonal antibody fragment.

Furthermore, the present invention is a method of making an antibody that binds at least two different human inhibitory KIR receptor gene products, said antibody expressing at least one of said two different human inhibitory KIR receptor gene products. In a population of NK cells that is capable of neutralizing the inhibition of NK cell cytotoxicity mediated by KIR, the following steps:
a) culturing the hybridoma of the present invention under conditions capable of producing the monoclonal antibody;
b) separating the monoclonal antibody from the hybridoma. Optionally, the method further comprises the step of producing a fragment of the monoclonal antibody, the step of producing a derivative of the monoclonal antibody, or the step of producing a derivative of such a monoclonal antibody fragment. Preferably, the antibody binds to a common determinant present on KIR2DL1 and KIR2DL2 / 3.

The present invention also provides a method of making an antibody that binds at least two different human inhibitory KIR receptor gene products, wherein the antibody expresses at least one of the two different human inhibitory KIR receptor gene products. In a population of NK cells that is capable of neutralizing the inhibition of NK cell cytotoxicity mediated by KIR, the following steps:
a) isolating the nucleic acid encoding the monoclonal antibody from the hybridoma of the present invention;
b) modifying the nucleic acid to an amino acid sequence corresponding to or substantially similar to the functional sequence of the monoclonal antibody (eg, at least about 65%, at least about 75%, at least about such sequence) An amino acid sequence that is about 85%, at least about 90%, at least about 95% (such as about 70 to 99%) identical, and is a humanized antibody, chimeric antibody, single chain antibody, immunoreactive fragment of an antibody, or Obtaining a modified nucleic acid comprising a sequence encoding a modified or derivatized antibody selected from a fusion protein comprising such an immunoreactive fragment, optionally performed;
c) when said nucleic acid or modified nucleic acid (ie a related nucleic acid encoding the same amino acid sequence) is inserted into an expression vector and said expression vector is present in a host cell grown under appropriate conditions, The encoded antibody or antibody fragment can be expressed; and
d) transfection of the expression vector into a host cell that does not produce immunoglobulin proteins if there is no transfection;
e) culturing the transfected host cell under conditions that cause expression of the antibody or antibody fragment;
f) isolating the antibody or antibody fragment produced by the transfected host cell. Preferably, the antibody binds to a common determinant present on KIR2DL1 and KIR2DL2 / 3.

Screening and selection of antibodies Certain antibodies of the invention inhibit KIR-mediated cytotoxicity of NK cells, specifically, inhibition mediated by the KIR2DL receptor, more specifically at least KIR2DL1 and KIR2DL2 / Inhibition by both 3-mediated inhibition can be reduced or neutralized. Thus, these antibodies, when interacting with MHC class I molecules, reduce, neutralize, and at least partially and detectably the inhibitory signaling pathways mediated by the KIR receptor, and An antibody that is “neutralizing”, “blocking” or “inhibitory” in the sense of blocking. More importantly, this neutralizing activity is responsible for some types of inhibitory KIR receptors, preferably some KIR2DL receptor genes, so that these antibodies can be used in most or all human subjects with high potency. It is expressed against the product, more preferably at least both KIR2DL1 and KIR2DL2 / 3. Neutralization of inhibition of cytotoxicity of NK cells mediated by KIR can be assessed by various assays or tests, such as the standard in vitro cytotoxicity assay described herein.

  Once an antibody that cross-reacts with multiple inhibitor KIR receptors is identified, the antibodies can be tested for the ability to reduce or neutralize the inhibitory effects of KIR receptors in intact NK cells. In certain variants, neutralizing activity can be expressed by the ability of the antibody to reconstitute lysis by target KIR2DL-expressing NK cells expressing HLA-C. In another specific embodiment, the neutralizing activity of the antibody is defined by the ability of the antibody to block or reduce binding of HLA-C molecules to KIR2DL1 and KIR2DL3 (or closely related KIR2DL2) receptors, and more preferably Binding to KIR2DL2 / 3 of an HLA-C molecule selected from Cw1, Cw3, Cw7 and Cw8 (or of another HLA-C molecule having an Asn residue at position 80) and / or Cw2, Defined as the ability of an antibody to alter the binding of a HLA-C molecule selected from Cw4, Cw5 and Cw6 (or another HLA-C molecule having a Lys residue at position 80) to KIR2DL1.

  In another variant, the neutralizing activity of the antibodies of the invention can be assessed in a cell-based cytotoxicity assay, as disclosed in the examples described herein.

In another variant, one HLA-C allele recognized by the test antibody and the KIR molecule of the NK population to stimulate cytokine production (eg, IFN-γ and / or GM-CSF production) of NK cells. The neutralizing activity of the antibody of the present invention can be evaluated in a cytokine release assay in which NK cells are incubated with a target cell line that expresses. In a typical protocol, IFN-γ production from PBMC is assessed by staining and analysis in the cell surface and cytoplasm by flow cytometry after about 4 days in culture. Briefly, Brefeldin A (Sigma Aldrich) can be added at a final concentration of about 5 μg / mL for at least about 4 hours of culture. Cells can then be incubated with anti-CD3 and anti-CD56 mAb prior to permeabilization (IntraPrep ^™ , Beckman Coulter) and staining with PE-anti-IFN-γ or PE-IgG1 (Pharmingen). GM-CSF and IFN-γ production from polyclonal activated NK cells was performed using an ELISA (GM-CSF: DuoSet Elisa, R & D Systems, Minneapolis, MN; IFN-γ: OptE1A set, Pharmingen) Can be measured in.

  The antibodies of the present invention may partially (ie reduce) or completely neutralize the inhibition of NK cell cytotoxicity mediated by KIR. For example, preferred antibodies of the invention are HLA-matched or HLA-compatible or autologous target cell populations (ie cell populations that would not be effectively lysed by NK cells in the absence of said antibody). Lysis can be induced or augmented. Thus, the antibodies of the invention can also be defined as promoting NK cell activity in vivo and / or in vitro.

  In certain embodiments, the antibody binds substantially the same epitope as monoclonal antibody DF200 (produced by hybridoma DF200), 1-7F9, or 1-4F1. Such antibodies are referred to herein as “DF200-like antibodies”, “1-7F9-like antibodies”, and “1-4F1-like antibodies”, respectively. In a further preferred embodiment, the antibody is a monoclonal antibody. More preferably, the “DF200-like antibody” of the present invention is an antibody other than the monoclonal antibody NKVSF1. Most preferred are monoclonal antibodies 1-7F9 or 1-4F1, and antibodies comprising the VH and / or VL regions of 1-7F9 or 1-4F1.

  Identification of one or more antibodies that bind to substantially or essentially the same epitope as the monoclonal antibodies described herein is one of a variety of immunological screening assays that can assess antibody competition. Any one can be easily determined. A number of such assays are commonly performed and well known in the art (see, eg, US Pat. No. 5,660,827, granted Aug. 26, 1997, incorporated herein by reference). Specifically incorporated). In order to identify an antibody that binds to the same or substantially the same epitope as the monoclonal antibody described herein, the actual determination of the epitope to which the antibody described herein binds is entirely It will be understood that it is not necessary.

  For example, if the test antibody to be tested is obtained from an animal from a different source or is an antibody of a different Ig isotype, a control antibody (eg, DF200) is mixed with the test antibody and KIR2DL1 and / or KIR2DL2 / 3 (both known to be bound by DF200) can be used to use a simple competition assay given to samples containing both or both. ELISA, radioimmunoassay, protocols based on Western blotting, and the use of BIACORE (eg, as described in the Examples section herein) are suitable for use in such simple competition studies.

  In certain embodiments, various amounts of test antibody (eg, about 1: 1, 1: 2, 1:10 or about 1: 100) are administered to a control antibody (eg, about 1: 1, 1: 2, 1:10 or about 1: 100) over a period of time before being given to the KIR antigen sample. For example, 1-7F9 or 1-4F1) will be premixed. In another embodiment, the control antibody and various amounts of test antibody can simply be added and mixed separately during exposure to the KIR antigen sample. As long as the bound antibody can be distinguished from the free antibody (eg, by using a separation or washing technique to remove unbound antibody) and the control antibody can be distinguished from the test antibody (eg, Whether the test antibody reduces binding of the control antibody to a different KIR2DL antigen (by using a species-specific or isotype-specific secondary antibody, or by a control antibody specifically labeled with a detectable label) Can be determined and would suggest that the test antibody recognizes substantially the same epitope as 1-7F9. Binding of (labeled) control antibody in the presence of a completely irrelevant antibody (which does not bind to KIR) can serve as a high value for the control. The low value of the control can be obtained by incubating the labeled control antibody with the same but unlabeled control antibody, in which case competition will occur and decrease the binding of the labeled antibody. In a test assay, the significantly decreased reactivity of the labeled antibody in the presence of the test antibody means that the test antibody recognizes substantially the same epitope (ie, competes with a labeled control antibody). It becomes an index. For example, 1-7F9: test antibody or 1-4F1: test antibody at any ratio between about 1: 1 or 1:10 to about 1: 100 with 1-7F9 or 1-4F1 KIR2DL1 and KIR2DL2 / 3 Any test antibody that reduces binding to the antigen by at least about 50%, such as at least about 60%, or more preferably at least about 70% (eg, about 65 to 100%) is 1-7F9 or It is considered an antibody that binds to substantially the same epitope or determinant as 1-4F1. Preferably, such a test antibody has a binding of 1-7F9 to at least one other (preferably each of the KIR2DL antigens) that has been observed in the absence of said test antibody as 1-7F9 or 1-4F1. Is preferably reduced to at least about 50%, at least about 60%, at least about 80%, or at least about 90% (eg, about 95%). Of course, such methods can be applied to identify and / or evaluate antibodies that compete with other antibodies and / or KIR antigens.

  Competition can be further or alternatively assessed, for example, by flow cytometry testing. In such a test, cells with a given KIR can be incubated first with a control antibody (eg 1-7F9 or 1-4F1) and then with a test antibody labeled with a fluorescent dye or biotin. . The binding obtained when preincubating with a saturating amount of control antibody is about 80% of the binding (measured using fluorescence) obtained with the test antibody without preincubation with the control antibody, preferably If about 50%, about 40% or less (eg, about 30%), the antibody is said to compete with the control antibody. Alternatively, binding obtained with a control antibody labeled (with a fluorescent dye or biotin) can be obtained without preincubation with the test antibody against cells preincubated with a saturating amount of test antibody. An antibody is said to compete with a control antibody if it is about 80%, preferably about 50%, about 40% or less (eg, about 30%) of the resulting binding.

  A simple competitive assay in which a test antibody is pre-adsorbed and applied at a saturating concentration to a surface on which either KIR2DL1 or KIR2DL2 / 3 or both are immobilized can also be used to advantage. The surface in a simple competition assay is preferably a BIACORE chip (or other medium suitable for surface plasmon resonance analysis). Binding of a control antibody (eg 1-7F9 or 1-4F1) to the KIR-coated surface was measured. The binding of the control antibody alone to the KIR containing surface is compared to the binding of the control antibody in the presence of the test antibody. In the presence of the test antibody, the test antibody is substantially identical to the control antibody so that the binding of the KIR2DL1 and KIR2DL2 / 3-containing surface by the control antibody is significantly reduced such that the test antibody “competes” with the control antibody. Recognize the epitope of. Reduce the binding of a control antibody (eg, 1-7F9 or 1-4F1) to both KIR2DL1 and KIR2DL2 / 3 antigen to at least about 20% or more, at least about 40%, at least about 50%, at least about 70% or more Any test antibody that is allowed to be considered can be an antibody that binds to substantially the same epitope or determinant as the control antibody (eg, 1-7F9 or 1-4F1). Preferably, such test antibody has at least about 50% (eg, at least about 60%, at least about 70% or more) binding of a control antibody (eg, 1-7F9 or 1-4F1) to each of the KIR2DL antigens. ) Will decrease. It will be appreciated that the order of the control antibody and the test antibody can be interchanged. That is, a control antibody can first be bound to a surface, and then a test antibody is then contacted with the surface in a competition assay. Since the degree of binding reduction seen for the second antibody (assuming the antibody is competing) is expected to be even greater, antibodies with higher affinity for the KIR2DL1 and KIR2DL2 / 3 antigens will first It is preferred to bind to surfaces containing KIR2DL1 and KIR2DL2 / 3. Additional examples of such assays include those described herein and, for example, “Saunal and Regenmortel (Saunal and Regenmortel, (1995) J. Immunol. Methods 183: 33-41), the contents of which are hereby incorporated by reference. Which is incorporated herein by reference).

  For purposes of illustration, although described for 1-7F9 or 1-4F1, the screening assay competes with one or more NKVSF1, DF200, EB6, GL183 and other antibodies, antibody fragments, and antibody derivatives according to the present invention. It will also be understood that it can be used to identify antibodies that do.

Once immunization and antibody production has been performed in a vertebrate or cell, specific selection steps can be performed as described in the claims to isolate the antibody. In this regard, in a specific embodiment, the present invention also relates to a method for producing such an antibody, the method comprising:
a) immunizing a non-human mammal with an immunogen comprising an inhibitory KIR2 polypeptide;
b) preparing an antibody that binds the KIR2DL polypeptide from the immunized animal;
c) selecting an antibody of (b) that cross-reacts with at least two different inhibitory KIR gene products;
d) can neutralize KIR-mediated inhibition of NK cell cytotoxicity against a population of NK cells expressing at least one of the two different human inhibitory KIR receptor gene products, (c) The step of selecting the antibody of).

  In certain embodiments, an additional step is performed between steps (c) and (d) above to select anti-KIR mAbs that do not react with KIR2DS4.

  Selection of antibodies that cross-react with at least two different inhibitory KIR gene products can be achieved by screening antibodies against two or more different inhibitory KIR antigens. In a further preferred embodiment, the antibody prepared in step (b) is a monoclonal antibody. Thus, as used herein, the term “preparing an antibody from the immunized animal” includes obtaining B cells from the immunized animal and producing a hybridoma that expresses the antibody. Using these B cells as well as obtaining antibodies directly from the serum of the immunized animal. In another preferred embodiment, the antibody selected in step (c) is an antibody that cross-reacts with at least KIR2DL1 and KIR2DL2 / 3.

  In yet another preferred embodiment, the antibody selected in step (d) is anti-KIR mAb as measured in a standard chromium release assay against target cells expressing cognate HLA class I molecules. At least about 10% more specific lysis mediated by NK cells exhibiting at least one KIR recognized by the antibody, preferably at least about It causes 40% more specific lysis, at least about 50% more specific lysis, or more preferably at least about 70% increased specific lysis (eg, about 60 to 100% increased specific lysis). Alternatively, it was used in a chromium assay using NK cell clones expressing one or several inhibitory KIRs and target cells expressing at least one HLA allele recognized by one of the KIRs on the NK clone. Sometimes the level of cytotoxicity obtained using said antibody is at least about 20% of the specific cytotoxicity obtained with the same effector: target cell ratio with the same concentration of DF200 or with blocking anti-MHC class I antibody. Preferably at least about 30% or more.

  The order of steps (c) and (d) of the method just described can be changed. Optionally, the method may include, in addition to or in place of the step, for example, generating a fragment of the monoclonal antibody or the monoclonal as described elsewhere herein. It may further comprise an additional step of making an antibody or derivative of such a fragment.

In another variant, the present invention further provides a method of obtaining an antibody, the method comprising:
(A) selecting from a library or repertoire a monoclonal antibody, a fragment of a monoclonal antibody, or a derivative of any of them, that cross-reacts with at least two different human inhibitory KIR2DL receptor gene products;
(B) can neutralize the inhibition of NK cell cytotoxicity mediated by KIR against a population of NK cells expressing said at least two different human inhibitory KIR2DL receptor gene products; Selecting an antibody, fragment or derivative.

  The repertoire can be any (recombinant) repertoire of antibodies or fragments thereof, optionally displayed by any suitable structure (eg, phage, bacteria, synthetic complex, etc.). Selection of inhibitory antibodies can be performed as disclosed above and as further described in the examples.

  Their published crystal structure (Maenaka et al. Structure with Folding and design 1999; 7: 391-398; Fan et al., Nature immunology 2001; 2: 452-460; Boyington et al. Nature, 2000; 405 : 537-543], the computer modeling of the extracellular domains of KIR2DL1, -2 and -3 (KIR2DL1-3) shows the interaction between these KIRs and the anti-KIR2DL cross-reactive mouse monoclonal antibodies DF200 and NKVSF1 , Specific regions or KIR2DL1, -2 and -3 were shown to be involved. Thus, in one embodiment, the present invention provides amino acid residues (105, 106, 107, 108, 109, 110, 111, 127, 129, 130, 131, 132, 133, 134, 135, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 181, and 192), an antibody that binds exclusively to KIR2DL1 is provided. In another embodiment, the present invention provides amino acid residues (105, 106, 107, 108, 109, 110, 111, 127, 129, 130, 131, 132, 133, 134, 135, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 181, and 192) antibodies that bind to KIR2DL1 and KIR2DL2 / 3 without interacting with amino acid residues outside the region defined by I will provide a.

In another embodiment, the invention relates to a variant of KIR2DL1 that binds to KIR2DL1 and wherein R131 (ie, the Arg residue at position 131 of the KIR2DL1 variant) is Ala (ie, substituted with Ala). Antibodies that do not bind are provided.
In another embodiment, the invention provides an antibody that binds to KIR2DL1 and does not bind to a variant of KIR2DL1 in which R157 is Ala.

  In another embodiment, the invention provides an antibody that binds to KIR2DL1 and does not bind to a variant of KIR2DL1 in which R158 is Ala.

  In another embodiment, the invention provides antibodies (131, 157, and 158) that bind to KIR2DL1 residues.

  In another embodiment, the invention provides an antibody that binds to KIR2DS3 (R131W) and does not bind to wild type KIR2DS3.

  In certain embodiments, the antibody is amino acid residues L38, R41, M44, F45, N46, D47, T48 according to the numbering system described in Wagtmann et al. (Wagmann et al., Immunity 1995; 2: 439-449). , L49, R50, I52, F64, D72, Y80, P87, and Y88 (characters representing amino acids are represented by a single letter, the number being the residue position in KIR2DL1 (SEQ ID NO: 23)) Binds to KIR2DL1 only within the region. These residues were identified as 1-7F9 KIR2DL1 epitopes (see Example 11).

  In another embodiment, the antibody binds to an antigenic epitope in a KIR2DL1 sequence consisting of a fragment from L38 to Y88 of SEQ ID NO: 23. In yet another embodiment, the antibody binds to an antigenic epitope in a KIR2DL1 sequence comprising a fragment from L38 to Y88 of SEQ ID NO: 23.

  In another embodiment, the present invention binds to KIR2DL1 and KIR2DL2 / 3 and provides residues L38, R41, M44, F45, N46, D47, T48, L49, R50, I52, F64, D72, Y80, P87, and Antibodies that do not interact with amino acid residues outside the region defined by Y88 are provided.

In another embodiment, the antibody comprises at least 10%, 20% of amino acid residues L38, R41, M44, F45, N46, D47, T48, L49, R50, I52, F64, D72, Y80, P87, and Y88, Binds to KIR2DL1 in the region containing 30%, 40%, 50%, 60%, 70%, 80%, 90% or more. In certain embodiments, the KIR2DL1 epitope for the antibody comprises amino acid residues M44 and F45 (see Examples 9 and 11).

  In another embodiment, the antibody comprises at least 1, 2, 4, amino acid residues L38, R41, M44, F45, N46, D47, T48, L49, R50, I52, F64, D72, Y80, P87, and Y88. Combines with KIR2DL1 in the region containing 6, 8, 10, or 12 or all. In certain embodiments, the region comprises amino acid residues M44 and F45, optionally L38, R41, N46, D47, T48, L49, R50, I52, F64, D72, Y80, P87, and Y88. Includes at least 1, 2, 4, 6, 8, 10 or all. In another specific embodiment, the region comprises at least amino acid residues M44, F45, and D72 in the KIR2DL1 region where the 1-7F9 epitope and the HLA-C binding site overlap.

  In another embodiment, the present invention provides antibodies that bind to KIR2DL1, KIR2DL2 / 3, and KIR2DS4.

  In another embodiment, the invention provides an antibody that binds to both KIR2DL1 and KIR2DL2 / 3, but does not bind to KIR2DS4.

  In another embodiment, the invention provides an antibody that binds to both KIR2DL1 and KIR2DL2 / 3, but does not bind to KIR2DS3 or to KIR2DS4.

The determination of whether an antibody, antibody fragment, or antibody derivative binds within one of the above defined epitope regions can be carried out by methods known to those skilled in the art. See, for example, Examples 9 and 11. In another example of such a mapping / characterization method, the epitope region for an anti-KIR antibody is by epitope “footprinting” using chemical modification of exposed amine / carboxyl in KIR2DL1 or KIR2DL2 / 3 protein. Can be determined. One specific example of such a footprinting technique is the use of HXMS (hydrogen-deuterium exchange, detected by mass spectrometry), in which the hydrogen / deuterium of the amide protons of the receptor and ligand proteins. Exchange, binding and reverse exchange occur and the backbone amide groups involved in protein binding will be protected from reverse exchange and will remain deuterated. At this point, the region of interest can be identified by proteolysis with pepsin, high performance small pore high performance liquid chromatography separation and / or electrospray ionization mass spectrometry. For example, Ehring [Ehring H, Analytical Biochemistry, Vol. 267 (2) pp. 252-259 (1999)] and / or Engen et al. [Engen, JR and Smith, DL (2001) Anal. Chem. 73, 256A-265A Please refer to]. Another example of a suitable epitope identification technique is nuclear magnetic resonance epitope mapping (NMR). In nuclear magnetic resonance epitope mapping (NMR), the location of signals in a two-dimensional NMR spectrum is typically compared for free antigen and antigen complexed with an antigen-binding peptide (such as an antibody). The antigen is typically selectively isotopically labeled with ¹⁵ N so that only the signal corresponding to the antigen is observed in the NMR spectrum and no signal from the antigen-binding peptide is observed. The antigen signal resulting from the amino acids involved in the interaction with the antigen-binding peptide is typically shifted in position in the complex spectrum relative to the free antigen spectrum, thus participating in binding. Amino acids can be identified. For example, the literature [Ernst Schering Res Found Worksho
p. 2004; (44): 149-67; Huang et al, Journal of Molecular Biology, Vol. 281 (1) pp. 61-67 (1998); and Saito and Patterson, Methods. 1996 Jun; 9 (3) : 516-24].

  Epitope mapping / identification can also be performed using mass spectrometry. See generally literature (Downward, J Mass Spectrom. 2000 Apr; 35 (4): 493-503 and Kiselar and Downard, Anal Chem. 1999 May 1; 71 (9): 1792-801).

  Protease digestion techniques may also be useful in epitope mapping and identification. Antigenic determinant related regions / sequences are obtained by using trypsin in a ratio of about 1:50 to KIR2DL1 or KIR2DL2 / 3 in protease digestion (eg, o / n digestion at 37 ° C. and pH 7-8). ) Followed by mass spectrometry (MS) for peptide identification. Peptides protected from trypsin cleavage by an anti-KIR antibody are subsequently incubated with a sample that has been trypsin digested with the antibody and then digested with, for example, trypsin (this reveals a footprint for the antibody). Can be identified. In similar epitope identification methods, other enzymes such as chymotrypsin, pepsin can be used, or other enzymes can be used instead. Furthermore, enzymatic digestion can provide a simple method for analyzing whether a potential antigenic determinant sequence is present in the region of KIR2DL1 for a KIR binding polypeptide. If the polypeptide is not exposed to the surface, it is likely that the immunogenicity / antigenicity is not relevant. See, for example, Manca (Manca, Ann Ist Super Sanita. 1991; 27 (1): 15-9) for a discussion of similar techniques.

  Site-directed mutagenesis is another technique useful for elucidating binding epitopes. For example, in “alanine scanning”, each residue in a protein segment was replaced with an alanine residue, and the results for binding affinity were measured. If the mutation induces significant resuction in binding affinity, it is likely to be involved in binding. Monoclonal antibodies specific for structural epitopes (ie, antibodies that do not bind to unfolded proteins) can be used to demonstrate that alanine substitutions do not affect the overall fold of the protein. See, for example, the literature (Clackson and Wells, Science 1995; 267: 383-386; and Wells, Proc Natl Acad Sci USA 1996; 93: 1-6).

  Electron microscopy can also be used for epitope “foot-printing”. For example, Wang et al. (Wang et al., Nature 1992; 355: 275-278) coordinated applications of cryoelectron microscopy, three-dimensional image reconstruction, and X-ray crystallography. Thus, the physical footprint of the Fab fragment on the capsid surface of the original cowpea mosaic virus was determined.

  Other forms of “label-free” assays for epitope assessment include surface plasmon resonance (SPR, BIACORE) and reflection interference spectroscopy (RifS). For example, the literature (Fagerstam et al., Journal Of Molecular Recognition 1990; 3: 208-14; Nice et al., J. Chromatogr. 1993; 646: 159-168; Leipert et al., Angew. Chem. Int. Ed 1998; 37: 3308-3311; Kroger et al., Biosensors and Bioelectronics 2002; 17: 937-944).

Antibody Fragments and Derivatives Antibodies of the present invention, preferably 1-7F9- or 1-4F1-like antibody fragments and derivatives (unless otherwise stated or clearly opposite, referred to as “antibody” as used herein) Can be made by techniques known in the art. “Immunoreactive fragments” comprise a portion of an intact antibody, generally the antigen binding site or variable region. Examples of antibody fragments include “kappa bodies” (see, eg, Ill et al., Protein Eng 10: 949-57 (1997)) and “janusins” (herein Fab, Fab ', Fab'-SH, F (ab) 2, F (ab') 2, dAb (Ward et al., (1989) Nature 341: 544-546); Included are fragments consisting essentially of VH domains), Fd (consisting essentially of VH and CH1 domains), and Fv (consisting essentially of VL and VH domains); diabodies; kappa bodies; and Janusin. For example, an antibody fragment includes a polypeptide having a primary structure consisting of one continuous sequence of consecutive amino residues (referred to herein as a “single-chain antibody fragment” or “single-chain polypeptide”. ), (1) a single chain Fv (scFv) molecule, (2) a single chain polypeptide containing only one light chain variable domain, or an associated heavy chain containing three CDRs of the light chain variable domain Those fragments without a portion, and (3) a single-chain polypeptide containing only one heavy chain variable region, or those containing three CDRs of a heavy chain variable domain and without an accompanying light chain portion As well as, but not limited to, as well as multispecific antibodies formed from antibody fragments. For example, Fab or F (ab ′) ₂ fragments can be generated by protease digestion of the isolated antibody according to conventional techniques.

  It will be appreciated that immunoreactive fragments can be modified using known methods, for example, to delay clearance in vivo and to obtain more desirable pharmacokinetic properties. For example, the fragment may be modified with polyethylene glycol (PEG) or polyoxyethylated polyol. Methods for linking PEG to Fab ′ fragments and conjugating site-specifically are described, for example, in “Leong et al, Cytokine 16 (3): 106-119 (2001) and Delgado et al, Br. J. Cancer 73 (2 ): 175-182 (1996), the disclosure of which is incorporated herein by reference.

  In certain aspects, the present invention provides antibodies, antibody fragments, and antibody derivatives comprising the 1-7F9 or 1-4F1 light and / or heavy chain variable region sequences shown in FIG. To do. In another aspect, the present invention relates to an antibody, antibody comprising one or more light and / or heavy chain variable region CDRs of 1-7F9 or 1-4F1 as shown in FIG. 15 or as described above Fragments and derivatives thereof are provided. Functional variants / analogs of such sequences are made by making appropriate substitutions, additions and / or deletions in these disclosed amino acid sequences using standard techniques. Sequence comparisons may be useful. From the superimposition of the crystal structures of 1-7F9 / KIR2DL1 and KIR2DL1 / MHC class I complexes (Fan et al., Nat. Immunol. 2001; 2: 452-460) (PDB-code 1IM9) It is concluded that scFv derivatives of 1-7F9 can block MHC class I binding to KIR2DL1. Also, a single L-chain of 1-7F9 or a single VL domain of 1-7F9 alone will block MHC class I binding when binding to KIR2DL1.

  Functional variants / analogs of such sequences are made by making appropriate substitutions, additions and / or deletions in these disclosed amino acid sequences using standard techniques. Sequence comparisons may be useful. In another aspect, positions where residues are present in the sequence of one of these antibodies, but where there are no residues in another antibody may be appropriate for deletions, substitutions and / or insertions.

  Alternatively, DNA encoding an antibody of the invention (eg, 1-7F9- or 1-4F1-like antibody) can be modified to encode a fragment of the invention. The modified DNA is then inserted into an expression vector and used to transform or transfect appropriate cells, as described further elsewhere herein, and then the cells are desired. The fragment of is expressed.

  In another embodiment, the DNA of the hybridoma producing the murine antibody (eg, DF-200-like antibody) of the invention is, for example, coding sequences for human heavy and light chain constant domains instead of homologous non-human sequences. [Morrison et al., Proc. Natl. Acad. Sci. USA, 81, pp. 6851 (1984)] can be modified prior to insertion into the expression vector. In another embodiment, the variable region encoding an antibody of the invention is linked by recombinant DNA technology to the entire immunoglobulin coding sequence or to a portion of the coding sequence for non-immunoglobulin polypeptides. May be. In this way, “chimeric” or “hybrid” antibodies are prepared that have the binding specificity of the original antibody. Typically, such non-immunoglobulin polypeptides are substituted for the constant domains of the antibodies of the invention.

Thus, according to another embodiment, an antibody of the invention, preferably a DF-200-like antibody, is humanized. “Humanized” forms of the antibodies of the invention include specific chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (Fv, Fab, Fab ′, F (ab ′) ₂ or other antigen-binding subsequences of antibodies, etc.). Typically, humanized antibodies are derived from the CDRs of the original antibody (donor antibody), while residues from the complementarity determining regions (CDRs) of the recipient maintain the desired specificity, affinity and ability of the original antibody. Is a human immunoglobulin (recipient antibody) that is replaced by In some cases, Fv framework residues of the human immunoglobulin can be replaced by corresponding non-human residues. Furthermore, humanized antibodies can comprise residues that are not found in the recipient antibody or any of the introduced CDR or framework sequences. These modifications are made to further refine and optimize antibody performance. In general, a humanized antibody will comprise substantially all of at least one, typically two variable domains. In a humanized antibody, all or substantially all CDR regions and all or substantially all FR regions corresponding to the CDR regions of the original antibody are of human immunoglobulin consensus sequence. A humanized antibody optimally will also typically comprise at least a portion of an immunoglobulin constant region (Fc) of a human immunoglobulin. For further details, see the literature [Jones et al., Nature, 321, pp. 522 (1986); Reichmann et al., Nature, 332, pp. 323 (1988); and Presta, Curr. Op. Struct. ., 2, pp. 593 (1992)].

  Methods for humanizing the antibodies of the present invention are well known in the art. Generally, the humanized antibody of the present invention has one or more amino acid residues introduced into the humanized antibody from the original antibody. These murine or other non-human amino acid residues are often referred to as “import” residues and are typically taken from an “import” variable domain. Humanization can be performed essentially according to the method of Winter and co-workers [Jones et al., Nature, 321, pp. 522 (1986); Riechmann et al., Nature, 332, pp. 323 (1988); Verhoeyen et al., Science, 239, pp. 1534 (1988)]. Thus, such “humanized” antibodies are chimeric antibodies in which the intact human variable domain is slightly replaced by the corresponding sequence from the original murine antibody (Cabilly et al., US Pat. No. 4,816,567). Indeed, humanized antibodies of the present invention typically have some CDR residues and possibly some FR residues replaced by residues from similar sites in the original mouse antibody. It is a human antibody.

  The choice of human variable domains to be used in the production of humanized antibodies (both light and heavy chain domains) is crucial to reducing antigenicity. According to the so-called “best-fit” method, the variable domain sequences of the antibodies of the invention are screened against the entire library of known human variable domain sequences. The human sequence closest to the mouse sequence is then accepted as the human framework (FR) for humanized antibodies [Sims et al., J. Immunol., 151, pp. 2296 (1993); Chothia and Lesk, J. Mol. Biol., 196, pp. 901 (1987)]. Another method uses a framework derived from the consensus sequence of all human antibodies of a particular subgroup of light or heavy chains. The same framework can be used for several different humanized antibodies (Carter et al., Proc. Natl. Acad. Sci. USA, 89, pp. 4285 (1992); Presta et al., J. Immunol., 51, pp. 1993)].

  Furthermore, it is important to humanize antibodies while retaining high affinity for multiple inhibitory KIR receptors and other favorable biological properties. To achieve this end goal, humanized antibodies are prepared by a process of analysis of the parental sequence and various conceptual humanized products, using a three-dimensional model of the parental and humanized sequence, according to a preferred method. . Three-dimensional immunoglobulin models are commonly available and are well known to those skilled in the art. Computer programs are available that draw and display the predicted three-dimensional structure of selected candidate immunoglobulin sequences. By examining these indications, it is possible to analyze the role that residues appear to play in the function of candidate immunoglobulin sequences, that is, to analyze residues that affect the ability of candidate immunoglobulins to bind to antigens. Become. In this way, FR residues can be selected and combined from the consensus and import sequences so that the desired antibody characteristic (such as increased affinity for the target antigen) is achieved. In general, CDR residues are directly and most heavily involved in influencing antigen binding.

As described above, methods for producing “humanized” monoclonal antibodies include using XenoMouse® (Abgenix, Fremont, Calif.). XenoMouse is a mouse host according to the present invention in which a mouse immunoglobulin gene is replaced by a functional human immunoglobulin gene. For this reason, the antibody produced by this mouse or the antibody produced in the hybridoma produced from the B cell of this mouse has already been humanized. XenoMouse is described in US Pat. No. 6,162,963, which is hereby incorporated by reference in its entirety. A similar method can be performed using HuMAb-Mouse ^™ (Medarex).

  Human antibodies can be obtained, for example, by using other transgenic animals engineered to express a human antibody repertoire for immunization [Jakobovitz et al., Nature 362 (1993) 255], or phage display methods. Can also be made according to a variety of other techniques, such as by selecting an antibody repertoire using. Such techniques are known to those skilled in the art and can be performed starting from the monoclonal antibodies disclosed herein.

  An antibody of the invention, preferably a 1-7F9- or 1-4F1-like antibody, is a “chimeric” antibody (immunoglobulin) (in a chimeric antibody, a portion of the heavy and / or light chain is present in the original antibody). May be derivatized to the same or homologous to the corresponding sequence, but the rest of the chain is from another species, or is identical or homologous to the corresponding sequence in an antibody belonging to another antibody class or subclass) It can be derivatized into a fragment of such an antibody as long as it exhibits the desired biological activity [Cabilly et al., Supra; Morrison et al., Proc. Natl. Acad. Sci. USA, 81, pp. 6851 (1984)].

Other derivatives within the scope of the present invention include functionalized antibodies, ie, toxins (such as ricin, diphtheria toxin, abrin and Pseudomonas exotoxins) or therapeutic radionuclides (eg, ⁹⁰ Y or ¹³¹ I); an antibody that is conjugated or covalently bound to a detectable moiety (such as a fluorescent moiety, a diagnostic radioisotope or a contrast agent); or a solid support (such as an agarose bead) included. Methods for conjugating or covalently binding these other factors to antibodies are well known in the art.

  Conjugation to toxins is useful for targeting and killing NK cells that present one of the cross-reactive KIR receptors on the cell surface. When the antibody of the present invention binds to the cell surface of such a cell, the antibody is taken inside and a toxin is released inside the cell to selectively kill the cell. Such use is another embodiment of the present invention.

  When the antibody of the present invention is used for diagnostic purposes, conjugation with a detectable moiety is useful. For such purposes, assaying a biological sample for the presence of NK cells having a cross-reactive KIR on its cell surface and detecting the presence of NK cells having a cross-reactive KIR in an organism. Is included, but is not limited thereto. Such assay and detection methods are also another embodiment of the present invention.

  Conjugation of the antibody of the present invention to a solid support is useful as a tool for affinity purification of NK cells having a cross-reactive KIR on the cell surface from a collection source such as a biological fluid. This purification method is another embodiment of the present invention, as is the resulting purified population of NK cells.

Pharmaceutical compositions and applications The present invention cross-reacts with at least two inhibitory KIR receptors present on the surface of NK cells to reduce or neutralize their inhibitory signals and enhance the activity of these cells A human or humanized antibody, or fragment and derivative thereof, in any suitable vehicle, in an amount effective to detectably enhance NK cell cytotoxicity in a patient or biological sample containing NK cells. A pharmaceutical composition is also provided. A pharmaceutical composition comprising a human or humanized anti-KIR mAb for use according to the present invention, if used with another active agent, if necessary, is a pharmaceutically acceptable carrier or diluent. Similarly, with any other known adjuvants and excipients, the prior art [eg, Remington (Remington: The Science and Practice of Pharmacy, 19th Edition, Gennaro, Ed., Mack Publishing Co., Easton, PA, 1995)]]. The composition may be in conventional forms such as capsules, tablets, aerosols, solvents or suspensions, or lyophilized powders.

  As described above, one object of the present invention is to provide a pharmaceutical formulation comprising a human or humanized antibody or fragment or derivative thereof, which is present at a concentration of 1 mg / ml to 500 mg / ml. And the formulation has a pH of 2.0 to 10.0. The formulation may further comprise a buffer system, a preservative, a tonicity agent, a chelating agent, a stabilizer and a surfactant. In one embodiment of the invention, the pharmaceutical formulation is an aqueous formulation (ie, a formulation comprising water). Such formulations are typically solutions or suspensions. In a further embodiment of the invention the pharmaceutical formulation is an aqueous solution. As used herein, the term “aqueous formulation” is a formulation that contains at least 50% w / w water. Similarly, the term “aqueous solution” is a solution containing at least 50% w / w water. The term “aqueous suspension” is a suspension containing at least 50% w / w water.

  In another embodiment, the pharmaceutical formulation is a freeze-dried formulation to which a solvent and / or diluent is added prior to use by a physician or patient.

  In another embodiment, the pharmaceutical formulation is a dry formulation (eg freeze-dried or spray-dried) ready for use without prior dissolution.

  In a further aspect, the present invention relates to a pharmaceutical formulation comprising an aqueous solution of the antibody and buffer described herein, wherein the antibody is present at a concentration of 1 mg / ml or greater and the formulation is about 2.0. Has a pH of ˜10.0.

  In another embodiment, the formulation has a pH of 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, Selected from the list consisting of 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, and 10.0.

  In a further embodiment, the buffer is sodium acetate, sodium carbonate, citrate, glycylglycine, histidine, glycine, lysine, arginine, sodium dihydrogen phosphate, disodium hydrogen phosphate ), Sodium phosphate, and tris (hydroxymethyl) -aminomethane, bicine, tricine, malic acid, succinate, maleic acid, fumaric acid, tartaric acid, aspartic acid or mixtures thereof. Each of these specific buffering agents constitutes an alternative aspect of the invention.

  In a further embodiment, the formulation further comprises a pharmaceutically acceptable preservative. In another embodiment, the preservative is phenol, o-cresol, m-cresol, p-cresol, methyl p-hydroxylbenzoate, propyl p-hydroxylbenzoate, 2-phenoxyethanol, butyl p-hydroxylbenzoate, 2 -Phenylethanol, benzyl alcohol, chlorobutanol, and thiomerosal, bronopol, benzoic acid, imidurea, chlorohexidine, sodium dehydroacetate, chlorocresol, ethyl p-hydroxylbenzoate, benzethonium chloride, chloro Selected from the group consisting of phenesin (3p-chlorophenoxypropane-1,2-diol) or mixtures thereof. In one embodiment, the preservative is present in a concentration from 0.1 mg / ml to 20 mg / ml. In another embodiment, the preservative is present in a concentration from 0.1 mg / ml to 5 mg / ml. In yet another embodiment, the preservative is present in a concentration from 5 mg / ml to 10 mg / ml. In yet another embodiment, the preservative is present in a concentration from 10 mg / ml to 20 mg / ml. Each of the specific preservatives listed constitutes an alternative aspect of the invention. The use of preservatives in pharmaceutical compositions is well known to those skilled in the art; see the literature [Remington: The Science and Practice of Pharmacy, 19th edition, 1995].

In a further embodiment, the formulation also includes an isotonic agent. In one embodiment, the tonicity agent is a salt (e.g., sodium chloride), sugar, sugar alcohol, amino sugar, amino acid (e.g., L-glycine, L-histidine, arginine, lysine, isoleucine, aspartic acid, tryptophan, Threonine), alditols (eg, glycerol (glycerin), 1,2-propanediol (propylene glycol), 1,3-propanediol, 1,3-butanediol), and polyethylene glycol (eg, PEG400), or mixtures thereof Selected from the group consisting of Any suitable sugar may be used, including but not limited to: mono-, di-, or polysaccharides; and water-soluble glucans, including fructose, including: Glucose, mannose, sorbose, xylose, maltose, lactose, sucrose, trehalose, dextran, pullulan, dextrin, cyclodextrin, soluble starch, hydroxyethyl starch and carboxymethylcellulose-Na. In one embodiment, the sugar additive is sucrose. Sugar alcohol is defined as a C4-C8 hydrocarbon having at least one -OH group, including, for example, mannitol, sorbitol, inositol, galactitol, dulcitol, xylitol, and arabitol. included. In one embodiment, the sugar alcohol additive is mannitol. The above sugars or sugar alcohols may be used individually or in combination. As long as the sugar or sugar alcohol is soluble in the liquid preparation and does not adversely affect the stabilizing effect achieved using the method of the present invention, there is no fixed limit on the amount used. In one embodiment, the sugar or sugar alcohol concentration is between about 1 mg / ml and about 150 mg / ml. In another embodiment, the tonicity agent is present in a concentration from 1 mg / ml to 50 mg / ml. In another embodiment, the tonicity agent is present in a concentration from 1 mg / ml to 7 mg / ml. In another embodiment, the tonicity agent is present in a concentration from 8 mg / ml to 24 mg / ml. In another embodiment, the tonicity agent is present in a concentration from 25 mg / ml to 50 mg / ml. Each of the specific tonicity agents in the above list constitutes an alternative aspect of the invention. The use of isotonic agents in pharmaceutical compositions is well known to those skilled in the art; see the literature [Remington: The Science and Practice of Pharmacy, 19th edition, 1995].

  In a further embodiment, the formulation also includes a chelating agent. In one embodiment, the chelating agent is selected from ethylenediaminetetraacetic acid (EDTA), citric acid, and aspartic acid salts, and mixtures thereof. In another embodiment, the chelator is present in a concentration from 0.1 mg / ml to 5 mg / ml. In another embodiment, the chelator is present in a concentration from 0.1 mg / ml to 2 mg / ml. In a further embodiment of the invention the chelating agent is present in a concentration from 2 mg / ml to 5 mg / ml. Each of these specific chelating agents constitutes an alternative aspect of the invention. The use of chelating agents in pharmaceutical compositions is well known to those skilled in the art. For convenience see the literature (Remington: The Science and Practice of Pharmacy, 19th edition, 1995).

  In a further embodiment, the formulation also includes a stabilizer. The use of stabilizers in pharmaceutical compositions is well known to those skilled in the art; see the literature [Remington: The Science and Practice of Pharmacy, 19th edition, 1995].

  The compositions of the present invention are, for example, stabilized liquid pharmaceutical compositions wherein the therapeutically active ingredient exhibits aggregate formation during storage in the liquid pharmaceutical formulation. It may be a stabilized liquid pharmaceutical composition comprising a potential polypeptide. “Aggregate formation” intends a physical interaction between polypeptide molecules that results in the formation of oligomers (which may remain soluble) or large visible aggregates precipitated from solution. “During storage” is intended to mean that a liquid pharmaceutical composition or formulation once prepared is not immediately administered to a subject. Rather, following preparation, it can be stored either in liquid form, frozen state, or dry form for later reconstitution into liquid form or other form suitable for administration to a subject. Packaged for. “Dried form” means that the liquid pharmaceutical composition or formulation is freeze-dried (ie, freeze-dried; see, eg, Williams and Polli (1984) J. Parenteral Sci. Technol. 38: 48-59). 18), Spray Drying (Masters (1991) in Spray-Drying Handbook (5th ed; Longman Scientific and Technical, Essez, UK), pp. 491-676; Broadhead et al. (1992) Drug Devel. Ind. Pharm. 18 : 1169-1206; and Mumenthaler et al. (1994) Pharm. Res. 11: 12-20) or air drying (Carpenter and Crowe (1988) Cryobiology 25: 459-470; and Roser (1991) Biopharm. 4: 47-53) is intended to be dried. Aggregate formation by a polypeptide during storage of a liquid pharmaceutical composition can adversely affect the biological activity of the polypeptide, resulting in a loss of therapeutic efficacy of the pharmaceutical composition. Furthermore, aggregate formation can cause other problems such as tubing, membrane, or pump occlusion when the polypeptide-containing pharmaceutical composition is administered using an infusion system.

  The pharmaceutical composition of the present invention may further comprise an amount of amino acid base sufficient to reduce aggregate formation by the polypeptide during storage of the composition. An “amino acid base” is an amino acid or amino acid in which any given amino acid exists in either its free base form or its salt form Is intended to be combined. When a combination of amino acids is used, all amino acids may be present in their free base form, all may be present in their salt form, or some are their free base While others may exist in the form of their salts. In one aspect, the amino acids used to prepare the compositions of the invention are those that retain a charged side chain such as, for example, arginine, lysine, aspartic acid, and glutamic acid. Any stereoisomer of the specific amino acid (methionine, histidine, imidazole, arginine, lysine, isoleucine, aspartic acid, tryptophan, threonine and mixtures thereof) (ie L or D isomers or mixtures thereof) or stereoisomers thereof Body combinations may be present in the pharmaceutical composition of the invention as long as the particular amino acid is present either in its free base form or in its salt form. In one embodiment, the L-stereoisomer is used. The compositions of the present invention may also be formulated with analogs of these amino acids. “Amino acid analogue” is intended to be a derivative of a natural amino acid that produces the desired effect of reducing aggregate formation by the polypeptide during storage of the liquid pharmaceutical composition of the invention. . Suitable arginine analogs include, for example, aminoguanidine, ornithine and N-monoethyl L-arginine. Suitable methionine analogs include ethionine and butionion, and suitable cysteine analogs. Includes S-methyl-L cysteine. As with other amino acids, amino acid analogs are introduced into the composition either in their free base form or in their salt form. In a further aspect of the invention, the amino acid or amino acid analog is used at a concentration sufficient to prevent or delay aggregation of the protein.

  In a further embodiment, a methionine (or other sulfur amino acid or amino acid analog) is a polypeptide comprising at least one methionine residue that is susceptible to subsequent oxidation, wherein said polypeptide acting as a therapeutic agent. In some cases, it may be added to inhibit oxidation of methionine residues to methionine sulfoxide. “Inhibit” intends the accumulation of methionine oxidized species to be minimal over time. By inhibiting methionine oxidation, the polypeptide will be better retained in its proper molecular form. Any stereoisomer of methionine (L or D) or any combination thereof can be used. The amount to be added should be sufficient to inhibit the oxidation of methionine residues and should be such that the amount of methionine sulfoxide is approved by regulatory agencies. Typically, this means that the composition contains about 10% to about 30% methionine sulfoxide or less. In general, this is accomplished by adding methionine such that the ratio of methionine added to the methionine residue ranges from about 1: 1 to about 1000: 1 (eg, 10: 1 to about 100: 1). Can be achieved.

  In a further embodiment, the composition also includes a stabilizer selected from the group consisting of high molecular weight polymers or low molecular compounds. In one embodiment, the stabilizer is polyethylene glycol (eg, PEG 3350), polyvinyl alcohol (PVA), polyvinyl pyrrolidone, carboxyl- / hydroxycellulose, or a derivative thereof (eg, HPC, HPC-SL, HPC- L and HPMC), sulfur-containing materials such as cyclodextrin, monothioglycerol, thioglycolic acid and 2-methylthioethanol, and different salts (eg sodium chloride). Each of these specific stabilizers constitutes an alternative aspect of the invention.

  The pharmaceutical composition also comprises additional stabilizers that further enhance the stability of the therapeutically active polypeptide therein. Stabilizers of particular importance to the present invention include methionine and EDTA (which protects the polypeptide against methionine oxidation), and nonionic surfactants (against aggregation associated with freeze thawing or mechanical shearing). Protects the polypeptide), but is not limited thereto.

In a further embodiment, the formulation also includes a surfactant. In one embodiment, the surfactant is a detergent, ethoxylated castor oil, polyglycolyzed glycerides, acetylated monoglyceride, sorbitan fatty acid ester, polyoxypropylene-polyoxyethylene block Polymers (e.g. poloxamers such as Pluronic® F68, poloxamers 188 and 407, Triton X-100), polyoxyethylene sorbitan fatty acid esters, polyoxyethylene and polyethylene derivatives (e.g. alkylated and alkoxylated derivatives (tweens , For example, Tween-20, Tween-40, Tween-80 and Brij-35), monoglycerides or ethoxylated derivatives thereof, diglycerides or polyoxyethylene derivatives thereof, alcohols, glycerol, lectins and phospholipids (e.g. phosphatidylse Phosphorus, phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, diphosphatidylglycerol and sphingomyelin), derivatives of phospholipids (e.g. dipalmitoylphosphatidic acid) and lysophospholipid derivatives (e.g. palmitoyllysophosphatidyl-L-serine and ethanolamine, 1-acyl-sn-glycero-3-phosphate ester of choline, serine or threonine) and alkyl, alkoxyl (alkyl ester), alkoxy (alkyl ether) -derivatives of lysophosphatidyl and phosphatidylcholine, such as lauroyl and myristoyl of lysophosphatidylcholine Derivatives, dipalmitoylphosphatidylcholine, and a modification of a polar head group, the head group comprising choline, ethanolamine, Sphatidic acid, serine, threonine, glycerol, inositol, and positively charged DODAC, DOTMA, DCP, BISHOP, lysophosphatidylserine and lysophosphatidylthreonine, and glycerophospholipids (e.g., cephalin), glyceroglycolipids (e.g., galactopyranoside) ), glycosphingolipid (eg, ceramides, gangliosides), dodecylphosphocholine, hen egg lysolecithin, fusidic acid derivatives - (e.g., tauro - sodium fusidic acid, etc.), long-chain fatty acids and salts thereof, C ₆ -C ₁₂ (eg , Oleic acid and caprylic acid), acylcarnitines and derivatives, Nα-acylated derivatives of lysine, arginine or histidine, or side-chain acylated derivatives of lysine or arginine, lysine, arginine or histidine and medium Sex or acid Nα-acylated derivatives of dipeptides with any combination of amino acids, Nα- of tripeptides with any combination of neutral amino acids and two charged amino acids Acylated derivatives, DSS (sodium docusate, CAS registration number [577-11-7]), calcium docusate, CAS registration number [128-49-4]), potassium docusate, CAS registration number [7491-09- 0]), SDS (sodium dodecyl sulfate or sodium lauryl sulfate), sodium caprylate, cholic acid or its derivatives, bile acids and their salts and glycine or taurine conjugates, ursodeoxycholic acid, sodium cholate, sodium deoxycholate , Sodium taurocholate, sodium glycocholate, N-hexadecyl-N, N-dimethyl-3-ammonio-1-propanesulfonate, anionic -Aryl-sulfonate) monovalent surfactant, zwitterionic surfactant (eg, N-alkyl-N, N-dimethylammonio-1-propanesulfonate, 3-cholamido-1-propyldimethyl) Ammonio-1-propanesulfonate, cationic surfactant (quaternary ammonium base) (eg cetyl-trimethylammonium bromide, cetylpyridinium chloride), nonionic surfactant (eg dodecyl β-D-glucopyranoside) ), Poloxamine (eg Tetronic's), which is a tetrafunctional block copolymer derived from the sequential addition of propylene oxide and ethylene oxide to ethylenediamine, or a surfactant that can be selected from the group consisting of imidazoline derivatives, or mixtures thereof , Selected from. Each of these specific surfactants constitutes an alternative aspect of the invention.

  The use of surfactants in pharmaceutical compositions is well known to those skilled in the art; see the literature [Remington: The Science and Practice of Pharmacy, 19th edition, 1995].

  In a further embodiment, the formulation also includes protease inhibitors such as EDTA (ethylenediaminetetraacetic acid) and benzamidine HCl, although other commercially available and suitable protease inhibitors may be used. The use of protease inhibitors is particularly useful in pharmaceutical compositions containing a protease zymogen to inhibit autocatalysis.

  Other components may also be present in the pharmaceutical formulation of the present invention. Such additional components include wetting agents, emulsifiers, antioxidants, bulking agents, tonicity modifiers, chelating agents, metal ions, oleaginous vehicles, proteins (eg, Human serum albumin, gelatin or protein) and zwitterions (eg amino acids such as betaine, taurine, arginine, glycine, lysine and histidine). Such additional components do not adversely affect the overall stability of the pharmaceutical formulation of the present invention.

  A pharmaceutical composition containing an antibody according to the present invention may be used to treat such treatment at several sites, such as local sites (eg skin and mucosal sites), sites that bypass absorption (eg in arteries, in veins). , Administration in the heart), may be administered to patients in need at the site where absorption is involved (eg, in the skin, under the skin, in muscle or in the abdomen).

  Administration of the pharmaceutical composition according to the present invention can be administered to patients in need of such treatment in the lingual, sublingual, buccal, in the mouth, oral, stomach and intestines ( in the stomach and intestine, nasal, lung (eg, via bronchiole and alveoli or a combination thereof), epidermal, dermal, transdermal, vaginal It may be via several routes of administration, such as, rectal, ocular (eg, via the conjunctiva), uretal, and parenteral.

  Compositions of the invention include solutions, suspensions, emulsions, microemulsions, complex emulsions, foams, salves, pastes, plasters, ointments, tablets, Coated tablets, rinses, capsules, eg hard and soft gelatin capsules, suppositories, rectal capsules, drops, gels, sprays, powders, aerosols, inhalants, ophthalmic solutions , Eye ointments, ophthalmic rinses, vaginal pessaries, vaginal rings, vaginal ointments, injection solutions, in situ transforming solutions such as in situ gelling agents, in situ hardeners (In situ setting), in situ precipitants, in situ crystallization agents, infusion solutions, and can be administered in several dosage forms such as implants.

  The composition of the present invention provides bioavailability that further enhances antibody stability to drug carriers, drug delivery systems and advanced drug delivery systems, for example, via covalent, hydrophobic and electrostatic interactions. Further formulated to increase-, increase solubility, reduce adverse effects, achieve chronotherapy well known to those skilled in the art, and increase patient compliance, or any combination thereof It may be compounded or attached. Examples of carriers, drug delivery systems and advanced drug delivery systems include polymers such as cellulose and derivatives, polysaccharides such as dextrans and derivatives, starches and derivatives, poly (vinyl alcohol), acrylate and methacrylate polymers , Polylactic acid and polyglycolic acid and block copolymers thereof, polyethylene glycol, carrier proteins such as albumin, gels, eg thermogelling systems, eg block block copolymer systems well known to those skilled in the art, micelles, liposomes, Microspheres, nanoparticulates, liquid crystals and their dispersants, L2 phase and its dispersants known to those skilled in the art of phase behavior in lipid-water systems, polymeric micelles, composites Marujon agents, self-emulsifying (self-emulsifying), self-microemulsifying, cyclodextrins and derivatives thereof, and include dendrimers (dendrimers) are not limited thereto.

  The composition of the present invention is used for pulmonary administration of antibodies using a metered dose inhaler, a dry powder inhaler, a nebulizer (all devices well known to those skilled in the art), and the like. Of solid, semisolids, powders and solutions.

  The compositions of the present invention are particularly useful for drug delivery systems that release in controlled, sustained, protracting, retarded, and slow. More specifically, but not limited to, the compositions are useful for the formulation of parenteral controlled release and sustained release systems (both systems lead to a multiple reduction in some administrations) well known to those skilled in the art. is there. Even more preferred are controlled release and sustained release systems administered subcutaneous. Without limiting the scope of the invention, examples of useful controlled release systems and compositions include hydrogels, oleaginous gels, liquid crystals, polymeric micelles, microspheres, Nanoparticles.

  Methods for producing a controlled release system useful in the composition of the invention include crystallization, condensation, co-crystallization, precipitation, co-precipitation, emulsification, dispersant, high pressure homogenisation. ), Encapsulation, spray drying, microencapsulating, coacervation, phase separation, microsphere production by solvent evaporation, extrusion and supercritical fluid processes processes), but is not limited to these. In general, pharmaceutical controlled release handbooks [Wise, DL, ed. Marcel Dekker, New York, 2000] and [Drug and the Pharmaceutical Sciences vol. 99: Protein Formulation and Delivery (MacNally, EJ, ed. Marcel Dekker). , New York, 2000].

  Parenteral administration may be performed by means of a syringe (optionally a pen-like syringe), with subcutaneous, intramuscular, intraperitoneal, or intravenous injection. Alternatively, parenteral administration can be performed by means of an infusion pump. A further option is a composition that is a solution or suspension for administering the antibody in the form of a nasal or pulmonal spray. As another option, the pharmaceutical composition containing the antibody can be administered transdermally (eg, by needle-free injection or from a patch, optionally from an iontophoretic patch). Or transmucosal (eg, buccal administration).

  The antibody can be administered via the pulmonary route using any known type of device suitable for pulmonary drug delivery as a solution, suspension or dry powder in a vehicle. Examples of these include three common types of aerosol generation for pulmonary drug delivery, including jet or ultrasonic nebulizers, metered dose inhalers, or dry powder inhalers (Yu J, Chien YW. Pulmonary drug delivery: Physiologic and mechanistic aspects. See, but not limited to, Crit Rev Ther Drug Carr Sys 14 (4) (1997) 395-453).

この関係への修正は、非球形粒子に関して発生する〔Edwards DA, Ben-Jebria A, Langer R. Recent advances in pulmonary drug delivery using large, porous inhaled particles. J Appl Physiol 84(2) (1998) 379-385を参照されたい〕。「MMAD」および「MMEAD」の用語は、当該技術に関して詳細に記載され、知られている〔Edwards DA, Ben-Jebria A, Langer R and represents a measure of the median value of an aerodynamic particle size distribution. Recent advances in pulmonary drug delivery using large, porous inhaled particles. J Appl Physiol 84(2) (1998) 379-385を参照されたい〕。空気動力学的中央粒子径（MMAD；Mass median aerodynamic diameter）および有効空気動力学的中央粒子径（MMEAD；mass median effective aerodynamic diameter）は、互換的に使用される統計的なパラメータであり、肺へ沈着する可能性に関しエアロゾル粒子のサイズを、実際の形状、サイズ、又は密度とは独立に経験的に記載する〔Edwards DA, Ben-Jebria A, Langer R. Recent advances in pulmonary drug delivery using large, porous inhaled particles. J Appl Physiol 84(2) (1998) 379-385を参照されたい〕。MMADは、インパクター（空気中での慣性性の行動を測定する機器）でなされる測定から通常計算される。 Based on standard test methods, the aerodynamic diameter (d _a ) of a particle is defined as the geometric equivalent diameter of the unit density (1 g / cm 3) of the reference standard spherical particle. In the simplest case, for spherical particles, d _a as a function of the square root of the density ratio as shown in formula, related to the reference diameter (d):

A correction to this relationship occurs for non-spherical particles [Edwards DA, Ben-Jebria A, Langer R. Recent advances in pulmonary drug delivery using large, porous inhaled particles. J Appl Physiol 84 (2) (1998) 379- See 385). The terms “MMAD” and “MMEAD” are well described and known in the art (Edwards DA, Ben-Jebria A, Langer R and represents a measure of the median value of an aerodynamic particle size distribution. Advances in pulmonary drug delivery using large, porous inhaled particles. See J Appl Physiol 84 (2) (1998) 379-385]. Aerodynamic median particle diameter (MMAD) and effective median aerodynamic diameter (MMEAD) are statistical parameters used interchangeably and to the lungs. Describe empirically the size of aerosol particles in relation to their potential for deposition, independent of actual shape, size or density [Edwards DA, Ben-Jebria A, Langer R. Recent advances in pulmonary drug delivery using large, porous inhaled particles. See J Appl Physiol 84 (2) (1998) 379-385]. MMAD is usually calculated from measurements made with an impactor (a device that measures inertial behavior in air).

  In further embodiments, the formulation is aerosolized by any known aerosol technique (eg, nebulisation) and is less than 10 μm (more preferably between 1 and 5 μm, most preferably between 1 and 3 μm). It would be possible to achieve MMAD of aerosol particles. The preferred particle size is based on the most suitable size for drug delivery to the deep lung, where protein is optimally absorbed [Edwards DA, Ben-Jebria A, Langer A, Recent advances in pulmonary drug delivery. using large, porous inhaled particles. See J Appl Physiol 84 (2) (1998) 379-385).

  Deposition of the pulmonary preparation containing the antibody into the deep lung can be accomplished by inhalation techniques [eg, slow inhalation flow (eg, 30 L / min), breath holding and timing of actuation (timing). can be further optimized by using a modification method of (but not limited to) of actuation).

  The term “stabilized formulation” means a formulation with increased physical stability, increased chemical stability or increased physical and chemical stability.

  As used herein, the term “physical stability” of protein formulations refers to the exposure of proteins to thermo-mechanical stresses and / or unstable interfaces ( interfaces) and the tendency of proteins to form biologically inert and / or insoluble aggregates of proteins as a result of interactions with surfaces (eg, hydrophobic surfaces and interfaces). The physical stability of an aqueous protein formulation is determined by exposing the formulation filled in a suitable container (e.g., cartridge or vial) to mechanical / physical stress (e.g., agitation) at different temperatures for different times. And then evaluated by means of visual inspection and / or turbidity measurement. Visual inspection of the formulation is performed in a sharp focused light with a dark background. The turbidity of the preparation corresponds to the degree of turbidity, for example, a scale of 0 to 3 (a preparation that does not show turbidity corresponds to a visual score of 0, and a preparation that shows visual turbidity in daylight is visually (Corresponding to a score of 3), characterized by visually scoring. If a formulation exhibits visual turbidity in daylight, it is classified as physically unstable with respect to protein aggregation. Alternatively, the turbidity of the formulation can be assessed by simple turbidity measurements well known to those skilled in the art. In addition, the physical stability of an aqueous protein preparation can be evaluated by using a spectroscopic drug or probe in a protein conformation state. The probe is preferably a small molecule that binds preferentially to a non-native conformer of the protein. An example of a small molecular spectroscopic probe of protein structure is Thioflavin T. Thioflavin T is a fluorescent dye that is widely used for the detection of amyloid fibrils. In the presence of fibrils (probably other protein configurations), Thioflavin T generates new maximum excitation at about 450 nm and enhanced emission at about 482 nm when bound to the fibril protein form. Unbound thioflavin T is essentially non-fluorescent at the wavelength.

  Other small molecules can be used as probes of changes in protein structure from natural to non-natural states. In one example, a “hydrophobic patch” probe preferentially binds to an exposed hydrophobic patch of protein. In general, hydrophobic patches are embedded within the protein's tertiary structure in the natural state, but are exposed when the protein begins to unfold or denature. Examples of these small molecule, spectroscopic probes are aromatic, hydrophobic dyes such as anthracene, acridine, phenanthroline and the like. Other spectroscopic probes are metal-amino acid complexes, such as cobalt metal complexes of hydrophobic amino acids such as phenylalanine, leucine, isoleucine, methionine, and valine.

  As used herein, the term “chemical stability” of a protein formulation is a chemical covalent change in protein structure, compared to the native protein structure, By changes that lead to the formation of chemical degradation products with potentially low biological potency and / or potentially increased immunogenic properties. Various chemical degradation products can be formed depending on the type and nature of the native protein and the environment to which the protein is exposed. The removal of chemical degradation is probably impossible to avoid completely. And an increase in the amount of chemical degradation products is often observed during storage and use of protein formulations, as is well known to those skilled in the art. Most proteins are prone to deamidation (a process in which side chain amide groups in glutaminyl or asparaginyl residues are hydrolyzed to form free carboxylic acids). Other degradation pathways involve the formation of high molecular weight transformation products. Here, two or more protein molecules are covalently linked to each other via transamidation and / or disulfide interactions, leading to the formation of covalent dimer, oligomer and polymer degradation products (Stability of Protein Pharmaceuticals , Ahern. TJ & Manning MC, Plenum Press, New York 1992). Oxidation (for example, of a methionine residue) can be mentioned as another variant of chemical degradation. The chemical stability of a protein formulation is that after exposure to different environmental conditions (degradation product formation can often be facilitated by increasing the temperature, to name one example) The amount can be assessed by measuring at various times. The amount of each individual degradation product can be determined by separating the degradation products using various chromatographic techniques (e.g., SEC-HPLC and / or RP-HPLC) depending on the molecular size and / or charge. Determined frequently.

  As outlined above, a “stabilized formulation” means a formulation with increased physical stability, increased chemical stability or increased physical and chemical stability. . In general, a formulation must be stable during use and storage (in compliance with recommended use and storage conditions) until the expiration date is reached.

  In one embodiment of the invention, the pharmaceutical formulation comprising the antibody is stable for more than 6 weeks of usage and for more than 3 years of storage.

  In another embodiment of the invention, the pharmaceutical formulation comprising the antibody is stable for more than 4 weeks of usage and for more than 3 years of storage.

  In a further embodiment of the invention the pharmaceutical formulation comprising the antibody is stable for more than 4 weeks of usage and for more than 2 years of storage.

  In yet a further embodiment of the invention the pharmaceutical formulation comprising the antibody is stable for more than 2 weeks of usage and for more than 2 years of storage.

  As noted above, pharmaceutically acceptable carriers that can be used in these compositions include ion exchangers; alumina; aluminum stearate; lecithin; serum proteins such as human serum albumin; phosphates and Buffer substances such as glycine; sorbic acid; potassium sorbate; partial glyceride mixtures of saturated plant fatty acids; water; salts or electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, and zinc salts; colloidal Silica; magnesium trisilicate; polyvinylpyrrolidone; cellulose-based material; polyethylene glycol; sodium carboxymethyl cellulose; polyacrylate; waxes; polyethylene-polyoxypropylene-block polymer; Le; and but are wool fat (wool fat) it is not intended to be limited thereto.

  The compositions of the present invention can be used in a method that enhances the activity of NK cells in a patient or biological sample. The method includes contacting the composition with the patient or biological sample. Such a method would be useful for both diagnostic and therapeutic purposes.

  When used with a biological sample, the antibody composition can be administered by simply mixing with or adding directly to the sample, depending on the nature of the sample (liquid or solid). The biological sample can be contacted directly with the antibody in any suitable device (plate, bag, flask, etc.). When used with a patient, the composition must be formulated for administration to the patient.

  As noted above, the compositions of the present invention can be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted container. The term “parenteral” as used herein includes subcutaneous, intravenous, intramuscular, intraarticular, intrasynovial, intrasternal, intrathecal, intrahepatic, intralesional and intracranial injection or infusion techniques. . Preferably, the composition is administered orally, intraperitoneally or intravenously.

  Sterile injectable forms of the compositions of this invention may be aqueous or oleaginous suspension. These suspensions may be formulated according to methods known in the art using suitable dispersing or wetting agents and suspending agents. A sterile injectable preparation may be a sterile injectable solution or solvent in a non-toxic parenterally acceptable diluent or solvent (eg, a solution in 1,3-butanediol) It may be a suspension. Among the acceptable vehicles and solvents that can be employed are water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile, fixed oils are conveniently employed as a solvent or suspending medium. For this purpose any bland fixed oil can be employed including synthetic mono- or diglycerides. Fatty acids such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are pharmaceutically acceptable natural oils such as olive oil or castor oil (especially in a polyoxyethylated manner). These oil solutions or suspensions can be long-chain alcohol diluents such as carboxymethyl cellulose or similar dispersants commonly used in the formulation of pharmaceutically acceptable dosage forms such as emulsions and suspensions or A dispersant may also be included. Other commonly used surfactants such as Tweens, Spans, and other emulsifiers or bioavailability commonly used in the manufacture of pharmaceutically acceptable solid, liquid or other dosage forms Other enhancers can also be used for formulation purposes.

  The compositions of the invention can be administered orally in any orally acceptable dosage form such as, but not limited to, capsules, tablets, aqueous suspensions or solutions. In the case of tablets for oral use, carriers that are commonly used include lactose and corn starch. Typically, a lubricant such as magnesium stearate is also added. For oral administration in a capsule form, useful diluents include lactose and dried corn starch. When aqueous suspensions are required for oral use, the active ingredient is mixed with emulsifying and suspending agents. If desired, certain sweetening, flavoring, or coloring agents can also be added.

  Alternatively, the compositions of the present invention can be administered in the form of suppositories for rectal administration. These should be prepared by mixing the drug with a suitable non-irritating excipient that is solid at room temperature but liquid at rectal temperature and can therefore dissolve and release the drug in the rectum. Can do. Such materials include cocoa butter, beeswax and polyethylene glycols.

  The compositions of the present invention can also be administered topically, especially when the target of treatment includes areas or organs that are easily accessible by topical application, such as diseases of the eye, skin or lower gastrointestinal tract. Appropriate topical formulations are readily prepared for each of these areas or organs.

  Topical application for the lower gastrointestinal tract can be effected with a rectal suppository formulation (see above) or a suitable enema formulation. Topically transdermal patches can also be used.

  For topical application, the compositions can be formulated in a suitable ointment containing the active component suspended or dissolved in one or more carriers. Carriers for topical administration of the compounds of this invention include, but are not limited to, mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene, polyoxypropylene compound, emulsifying wax and water. Absent. Alternatively, the composition can be formulated in a suitable lotion or cream containing the active ingredient suspended or dissolved in one or more pharmaceutically acceptable carriers. Suitable carriers include, but are not limited to, mineral oil, sorbitan monostearic acid, polysorbate 60, cetyl ester wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water.

  For ophthalmic use, the composition is as a finely divided suspension in isotonic sterile physiological salt adjusted for pH, or preferably with a preservative such as benzylalkonium chloride added, Alternatively, it can be formulated as a solution in sterile pH adjusted isotonic physiological saline. Alternatively, for ophthalmic applications, the composition can be formulated in an ointment such as petrolatum.

  The compositions of the invention can also be administered by nasal aerosol or inhalation. Such compositions are prepared according to techniques well known in the pharmaceutical formulation art and include benzyl alcohol or other suitable preservatives, absorption enhancers to enhance bioavailability, carbon perfluoride and / or Alternatively, it can be prepared as a solution in physiological saline using conventional solubilizing or dispersing agents.

Several monoclonal antibodies have been shown to be effective in the clinical setting, such as Rituxan (Rituximab), Herceptin (Trastuzumab) or Xolair (Omalizumab), and similar dosage regimens (ie, formulation and / or dosage and (Or administration protocol) can be used with the antibodies of the invention. Schedules and dosages for administration of antibodies in the pharmaceutical compositions of the invention can be determined according to methods known for these products using, for example, manufacturer's instructions. For example, the antibody present in the pharmaceutical composition of the invention can be provided in a 100 mg (10 mL) or 500 mg (50 mL) disposable vial at a concentration of 10 mg / mL. The product is formulated in 9.0 mg / mL sodium chloride, 7.35 mg / mL sodium citrate dihydrate, 0.7 mg / mL polysorbate 80, and sterile water for injection for intravenous administration. The Adjust the pH to 6.5. A typical suitable dosage range for an antibody in a pharmaceutical composition of the invention can be between about 10 mg / m ² and 500 mg / m ² . However, these schedules are typical and the optimal schedule is considered, taking into account the affinity and tolerability of the specific antibodies in the pharmaceutical composition, which must be determined in clinical trials. It will be understood that the dosage regimen can be adapted. The infusion volume and schedule of antibodies in the pharmaceutical composition of the present invention that saturate NK cells for 24 hours, 48 hours, 72 hours or one week or one month is the amount of antibody in human and non-human mammals. It will be determined taking into account the affinity and pharmacokinetic parameters of the antibody.

  According to another embodiment, the antibody composition of the invention may further comprise another therapeutic agent, including an agent normally used for the specific therapeutic purpose for which the antibody is administered. The additional therapeutic agent will usually be present in the composition in a monotherapy for the disease or condition being treated, in an amount typically used for that therapeutic agent. Such therapeutic agents include therapeutic agents used to treat cancer, therapeutic agents used to treat infectious diseases, therapeutic agents used for other immunotherapy, cytokines (IL-2 or IL -15)), and other antibodies and fragments of other antibodies, but are not limited thereto.

  In another embodiment, against NK cells that bind common determinants present on at least two different human inhibitory KIR receptor gene products and express at least one of said two different human inhibitory KIR receptors. Delivery of antibodies of the invention capable of neutralizing KIR-mediated inhibition of NK cell cytotoxicity, alone or induced to tumor or inflammatory sites in humans and other non-human mammals Together with another substance to do so can be incorporated into liposomes (immunoliposomes). Such other substances include nucleic acids for delivering genes for gene therapy; or nucleic acids for delivering antisense RNA, RNAi or siRNA for suppressing genes in NK cells; or NK cells Toxins or drugs to target and kill.

  For example, a number of therapeutic agents can be used for the treatment of cancer. The antibody compositions and methods of the present invention can be combined with any other method commonly used in the treatment of specific diseases, particularly tumors, cancer diseases, or other diseases or disorders that a patient develops. . Unless a therapeutic approach is known per se to be detrimental to patient symptoms and does not significantly negate the activity of the antibody in the pharmaceutical composition of the invention, the combination with the present invention Is assumed.

  With respect to the treatment of solid tumors, the pharmaceutical compositions of the present invention can be used with classic approaches such as surgery, radiation therapy, chemotherapy and the like. Thus, the present invention uses the pharmaceutical composition of the present invention at the same time as surgery or radiation therapy, before or after surgery or radiation therapy; or conventional chemotherapy, radiation therapy or anti-angiogenic agent, or target A combination therapy is provided that is administered to a patient before or after them with an immunotoxin or coaguligand induced in

  In the treatment plan, when one or more drugs are used together with the antibody-containing composition of the present invention, the combined result is the sum of the effects observed when each treatment is performed separately. There is no need. At least an additive effect is generally desirable, but any increased anticancer effect over one of the monotherapy would be beneficial. In addition, it is possible and advantageous to exert a synergistic effect, but it is not necessary for the combination treatment to exert a synergistic effect.

  In order to carry out a combined anticancer therapy, the antibody composition of the present invention is simply administered to a patient together with another anticancer agent in a manner effective to bring the combined anticancer effect in an animal. Would be good. Thus, the agents will be given in an amount and duration that is effective to cause them to coexist in the tumor vasculature and produce a combined effect in the tumor environment. To achieve this goal, the antibody composition and anticancer agent of the invention are administered to a patient simultaneously in a single combination composition or as two separate compositions using the same or different routes of administration. obtain.

  Alternatively, administration of the antibody composition of the present invention can be performed before or after anticancer drug treatment, for example, at intervals ranging from minutes to weeks and months. It will ensure that the anti-cancer agent and the antibody in the antibody composition of the present invention advantageously exert a combined effect against cancer. By way of example, the mAbs of the invention can be administered to patients with non-Hodgkin lymphoma (NHL). Such patients are typically treated in combination with Rituximab and a chemotherapeutic agent known as CHOP. Thus, using the anti-KIR antibodies of the present invention, NHL patients experiencing treatment with rituximab and CHOP are all treated on a treatment schedule where the drug is given on the same day or on a different day for a longer treatment period. Can be treated by combining the administration of different drugs.

  Other anti-cancer agents may be given prior to, simultaneously with, or following administration of the anti-KIR antibody composition of the invention. However, when an antibody immunoconjugate is used in the antibody composition of the present invention, various anticancer agents can be administered simultaneously or sequentially.

  In some situations, it may even be desirable to significantly extend the period for treatment, in which case several days (between each administration of the anti-cancer agent or anti-cancer treatment and the antibody composition of the invention) 2, 3, 4, 5, 6 or 7), weeks (1, 2, 3, 4, 5, 6, 7 or 8) or months (1, 2, 3, 4, 5, 6, 7) Or 8) elapses. This is because anti-cancer treatment is intended to substantially destroy the tumor, and the administration of the antibody composition of the present invention is intended to suppress micrometastasis or tumor regrowth (such as surgery or chemotherapy) ) Would be advantageous.

  It is also envisaged that any of the anti-KIR antibody-based compositions or anticancer agents of the present invention will be administered more than once. These agents may be administered alternately every other day or every other week, or after repeated treatment with the anti-KIR antibody composition of the present invention, anticancer drug therapy may be repeated. In any case, what is needed to achieve tumor regression using combination therapy is a combined amount effective to exert an anti-tumor effect, regardless of the number of doses, both drugs Only to deliver.

  With regard to surgery, any surgical intervention can be performed in conjunction with the present invention. For radiation therapy, any mechanism for inducing DNA damage locally in cancer cells, such as gamma radiation, X-rays, UV radiation, microwaves, and even electron radiation, is envisioned. It is also envisaged to induce and deliver radioisotopes to cancer cells, which can be used in combination with target-inducing antibodies or other target-inducing means.

  In other aspects, an immunomodulatory compound or therapy can be administered in combination with or as part of an antibody composition of the invention. Preferred examples of immunomodulatory compounds include cytokines. Various cytokines can be used in such a combined approach. Examples of cytokines useful in the above combinations envisioned by the present invention include IL-1alpha IL-1beta, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL -8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-15, IL-21, TGF-beta, GM-CSF, M-CSF, G-CSF, TNF-alpha , TNF-beta, LAF, TCGF, BCGF, TRF, BAF, BDG, MP, LIF, OSM, TMF, PDGF, IFN-alpha, IFN-beta, IFN-gamma. Cytokines used in the combination therapy or composition of the invention are administered according to a standard dosing regimen, tailored to clinical indicators such as patient symptoms and relative toxicity of the cytokines. Other immunomodulatory compounds that can be administered in combination with or as part of the antibody composition of the present invention include anti-CTLA4 antibodies or anti-CD94 / NKG2A antibodies (eg, US Published Patent Publications). And antibodies that specifically bind to other inhibitory receptors on lymphocytes, including but not limited to antibodies such as: 20030095965). Variants and derivatives of these molecules known in the art can be used additionally or alternatively in such methods and suitably introduced into the compositions of the present invention.

  In some embodiments, therapeutic compositions of the invention comprising cross-reactive, blocking, and / or inhibitory anti-KIR antibodies can be administered with a chemotherapeutic or hormonal therapeutic agent, or chemotherapy An agent or hormonal therapeutic agent can further be included. A variety of hormonal and chemotherapeutic agents can be used in the combination therapy disclosed herein. Examples of chemotherapeutic agents envisaged as typical include alkylating agents, antimetabolites, cytotoxic antibiotics, vinca alkaloids such as adriamycin, dactinomycin, mitotic machine, carminomycin, daunomycin, doxorubicin, Tamoxifen, taxol, taxotere, vincristine, vinblastine, vinorelbine, etoposide (VP-16), 5-fluorouracil (5FU), cytosine arabinoside, cyclophosphamide, thiotepa, methotrexate, camptothecin, actinomycin-D, mitomycin C, cisplatin CDDP), aminopterin, combretastatin, and derivatives and prodrugs thereof, but are not limited thereto.

  Hormonal agents include, for example, LHRH agonists such as leuprorelin, goserelin, triptorelin and buserelin; antiestrogens such as tamoxifen and toremifene; antiandrogens such as flutamide, nilutamide, cyproterone and bicalutamide; anastrozole, exemestane, letrozole and Aromatase inhibitors such as fadrozole; and progesterone hormones such as, but not limited to, medroxy, chlormadinone and megestrol.

Appropriate doses of chemotherapeutic agents are already used in clinical therapy, where the chemotherapeutic agent is administered alone or in combination with other chemotherapeutic agents, as will be apparent to those skilled in the art. It will be close to the dose. By way of example only, drugs such as cisplatin and other DNA alkylating agents can be used. Cisplatin is widely used to treat cancer, and the effective dose used in clinical applications is 20 mg / m ² every 3 weeks for 5 days over a total of 3 therapeutic units. Cisplatin is not absorbed orally and must therefore be delivered by intravenous, subcutaneous, intratumoral or intraperitoneal injection.

  Additional useful chemotherapeutic agents include compounds that interfere with DNA replication, mitosis and chromosomal segregation, and agents that disrupt the synthesis and fidelity of polynucleotide precursors. A number of typical chemotherapeutic agents for combination therapy are listed in Table C of US Pat. No. 6,524,583, the disclosure of drugs and indications is expressly incorporated herein by reference. Incorporated. Each of the listed drugs is exemplary and not limiting. Those skilled in the art are referred to the literature ["Remington's Pharmaceutical Sciences" 15th Edition, chapter 33, in particular pages 624-652]. Variations in dosage may occur depending on the condition being treated. The treating physician will be able to determine the appropriate dose for each subject.

  The cross-reactive, blocking, and / or inhibitory anti-KIR antibody compositions of the present invention can be used with any one or more anti-angiogenic therapies or can further comprise an anti-angiogenic agent. Examples of such agents include neutralizing antibodies, antisense RNA, siRNA, RNAi, RNA aptamers and ribozymes that are each induced against VEGF or the VEGF receptor (US Pat. No. 6,524,583). The disclosure of which is incorporated herein by reference). Variants of VEGF that have antagonist properties can also be used, as described in WO 98/16551, which is expressly incorporated herein by reference. Exemplary anti-angiogenic agents for use with combination therapy are further listed in Table D of US Pat. No. 6,524,583, the disclosure of drugs and indications is incorporated herein by reference. Clearly incorporated.

  The anti-KIR antibody compositions of the present invention can be advantageously used in combination with a method for inducing apoptosis or can include an apoptotic agent. For example, a number of oncogenes that inhibit apoptosis (ie, programmed cell death) have been identified. Exemplary oncogenes belonging to this category include bcr-abl, bcl-2 (bc-1, different from cyclin D1; GenBank accession numbers M14745, X06487; US Pat. No. 5,650,491; , 539,094, each incorporated herein by reference), including but not limited to family members including Bcl-x1, Mcl-1, Bak, A1 and A20. Overexpression of bcl-2 was first discovered in T cell lymphoma. The oncogene bcl-2 functions by binding to Bax (a protein in the apoptotic pathway) and inactivating Bax. Inhibition of bcl-2 function suppresses Bax inactivation and advances the apoptotic pathway. Inhibition of this class of oncogenes is envisioned for use in the present invention to obtain enhanced apoptosis using, for example, antisense nucleotide sequences, RNAi, siRNA, or small molecule chemical compounds (US Patent No. 5,650,491; 5,539,094; and 5,583,034, each incorporated herein by reference).

  The anti-KIR antibody compositions of the invention comprise a molecule (eg, an antibody, ligand, or conjugate thereof) that is directed against a specific marker in a target cell (eg, a target tumor cell). “Target inducers”) can also be included or used with the molecule. Generally speaking, targeted inducers for use in such additional aspects of the invention are preferably accessible tumors that are preferentially or specifically expressed at the tumor site. It will be preferred to recognize the antigen. In general, a target inducer will bind to a component of a tumor cell expressed on the surface, accessible to the surface, or localized to the surface. The target inducer also preferably exhibits high affinity properties, such as heart, kidney, brain, liver, bone marrow, large intestine, breast, prostate, thyroid, gallbladder, lung, adrenal gland, muscle, nerve fiber, pancreas, skin, or It would be preferable not to exert significant side effects in vivo against normal tissues that support life, such as one or more tissues selected from other organs or tissues that support life in the human body. As used herein, the term “does not exert significant side effects” means that when administered in vivo, the target inducer is negligible or clinical in terms of side effects normally encountered during chemotherapy. Represents facts that can only occur in a manageable manner.

  In treating tumors, the antibody composition of the present invention can further comprise an auxiliary compound or can be used in conjunction with the auxiliary compound. Auxiliary compounds include, by way of example, antiemetics such as serotonin antagonists, and treatment of phenothiazines, substituted benzamides, antihistamines, butyrophenones, corticosteroids, benzodiazepines and cannabinoids; bisphosphonates such as zoledronic acid and pamidronic acid And hematopoietic growth factors such as erythropoietin and G-CSF, such as filgrastim, lenograstim and darbepoetin.

  In another embodiment, two or more antibodies of the invention (NKVSF1, DF200, 1) that recognize different epitopes or determinants to reduce or neutralize the inhibitory effects of as many KIR gene products as possible. -4F1 and / or 1-7F9) can be combined in a single composition. A composition comprising a cross-reactive inhibitory KIR antibody of the present invention or a combination of fragments or derivatives thereof, a small percentage that may lack each inhibitory KIR gene product recognized by a single cross-reactive antibody. It may be possible to use it even more widely since there may be a human population. Similarly, the antibody composition of the present invention may further comprise one or more antibodies that recognize a single inhibitory KIR subtype. Such a combination would also provide wider use in therapeutic settings. Thus, the antibodies of the invention can be combined with another anti-KIR antibody that binds to one or more KIR2DL1, KIR2DLK2, KIR2DL3, KIR3DL1, KIR3DL2, and KIR3DL3, and the like.

  The present invention also provides a method of enhancing NK cell activity in said patient comprising the step of administering the composition of the present invention to a patient in need of enhancing NK cell activity. The method is more particularly beneficial for increased NK cell activity, caused by, affected by, or caused by cells susceptible to lysis by NK cells, or by insufficient NK cell activity. It is directed to increasing NK cell activity in patients who have a disease caused by or insufficient NK cell activity (such as cancer, other proliferative diseases, infectious diseases or immune diseases). More specifically, the methods of the invention include carcinomas (including bladder, breast, colon, kidney, liver, lung, ovary, prostate, pancreas, stomach, neck, thyroid and skin carcinomas, including squamous cell carcinoma). Hematopoietic tumors of the lymphatic system (including leukemia, acute lymphoblastic leukemia, acute lymphoblastic leukemia, B cell lymphoma, T cell lymphoma, Hodgkin lymphoma, non-Hodgkin lymphoma, hair cell lymphoma, and Burkitts lymphoma) ); Hematopoietic tumors of myeloid lineage (including acute and chronic myeloid leukemia and promyelocytic leukemia); tumors of mesenchymal origin (including fibrosarcoma and rhabdomyosarcoma); other tumors (melanoma, Seminoma, teratocarcinoma, neuroblastoma and glioma); tumors of the central and peripheral nervous system (including astrocytoma, neuroblastoma, glioma and schwannomas); Tumors of mesenchymal origin (fiber Including sarcomas, rhabdomyosarcomas and osteosarcomas); and other tumors (including melanoma, xeroderma pigmentosum, keratophyte celloma, seminoma, thyroid follicular carcinoma and teratocarcinoma) Used for the treatment of various cancers and other proliferative diseases.

  Other preferred diseases that can be treated according to the present invention include hematopoietic tumors of the lymphoid lineage (eg, T prolymphocytic leukemia (T-PLL), including small cell and brain cell types); Spherical leukemia (LGL), preferably of the T cell type; Sezary syndrome (SS); adult T cell leukemia lymphoma (ATLL); a / d T-NHL hepatosplenic lymphoma; And immunoblastic subtype); angioimmunoblastic T cell lymphoma; angiocentric (nasal) T cell lymphoma; undifferentiated (Ki 1+) large cell lymphoma; intestinal T cell lymphoma; T lymphoblast type; T cell and B cell tumors, including but not limited to T cell diseases such as lymphoma / leukemia (T-Lbly / T-ALL).

  For example, hyperplasia, fibrosis (especially other types of fibrosis such as pulmonary fibrosis, renal fibrosis), angiogenesis, psoriasis, atherosclerosis, and smooth muscle proliferation in blood vessels (stenosis or post-angioplasty or Other proliferative diseases, including restenosis) can also be treated according to the present invention. The cross-reactive inhibitory KIR antibodies of the present invention can be used to treat or prevent infectious diseases, including any infection preferably caused by a virus, bacteria, protozoan, mold or fungus. Such viral infectious organisms include hepatitis A, hepatitis B, hepatitis C, influenza, chickenpox, adenovirus, herpes simplex virus I (HSV-1), herpes simplex virus 2 (HSV-1), Rinderpest, rhinovirus, echovirus, rotavirus, respiratory syncytial virus, papillomavirus, papillomavirus, cytomegalovirus, echinovirus, arbovirus, hantavirus, coxsackie virus, mumps virus, measles Virus, rubella virus, poliovirus, Ebola virus, and human immunodeficiency syndrome virus type I or type 2 (HIV-1, HIV-2), but are not limited to these.

  Bacterial infections that can be treated according to the present invention include: Staphylococcus; Streptococcus (including S. yogenes); Enterococci; Bacillus ( Bacillus anthracis and Lactobacillus); Listeria; Corynebacterium diphtheriae; Gardnerella (including G. vaginalis); (Thermoactinomycesvulgaris); Trepone Treponema; Campylobacter, Pseudomonas (including Raeruginosa); Legionella; Neisseria (N. gonorrhoea and N. gonorrhoea; .. meningosepeticum and F. odoraturn); Brucella; Bordetella (including B. pertussis and B. bronchiseptica); Escherichia (including E. boli); Enterobacter (E nterobacter, Serratia (including S. marcescens and S. lifacfaciens); Edwardsiella; Proteus (included P. mirabilis and P. vulgaris; Rickettsiaceae (including R. fickettsfi), Chlamydia (including C. psittaci and C. trachorantis); Mycobacterium (M. tuberculis, M. .Laprae M. avium, M.M. bovis, M.M. africanum, M.M. Kansasii, M.M. intracellulare and M.C. including, but not limited to, infections caused by Nocardia).

  Protozoal infections that can be treated according to the present invention include, but are not limited to, infections caused by Leishmania, kokuzidioa and trypanosoma. A complete list of infectious diseases can be found on the National Center for Infectious Diseases (NCID) at the Centers for Disease Control (CDC) (World Wide Web (www) at cdc.gov/ncidod/diseases/) This list is incorporated herein by reference. All of the above diseases are candidates for treatment with the cross-reactive inhibitory KIR antibody of the present invention.

  Such methods of treating various infectious diseases can be used alone or in other treatments and / or therapeutic agents known to treat such diseases (antiviral agents, antifungal agents, antibacterial agents). In combination with antibiotics, antiparasitic agents and antiprotozoal agents) can be used. Where these methods involve additional treatment with additional therapeutic agents, these therapeutic agents may be administered with the antibodies of the invention as a single dosage form or as separate multiple dosage forms. it can. When administered as a separate dosage form, the additional therapeutic agent can be administered before, simultaneously with, or after administration of the antibody of the invention.

  Further aspects and advantages of the present invention are disclosed in the experimental part below. The following experimental part is exemplary and should not be construed to limit the scope of the present application.

[Example]
Example 1: Purification of PBLs and generation of polyclonal or clonal NK cell lines
PBLs were obtained from healthy donors by depleting Ficoll-Hypaque gradients and plastic adherent cells. To obtain enriched NK cells, PBLs were incubated with anti-CD3, anti-CD4 and anti-HLA-DR mAb (30 minutes at 4 ° C), then goat anti-mouse magnetic beads (Dynal) (30 minutes at 4 ° C) ) And immunomagnetic selection was performed by methods known in the art (Pende et al., J Exp Med 1999; 190: 1505-1516). Culturing CD3-, CD4-, and DR-cells with irradiated feeder cells, 100 U / mL interleukin 2 (Proleukin, Chiron Corporation), and 1.5 ng / mL phytohemagglutinin A (GibcoBRL) Obtained. NK cells were cloned by limiting dilution and NK cell clones were characterized by flow cytometry for expression of cell surface receptors.

  The mAbs used were JT3A (IgG2a, anti-CD3), EB6 and GL183 (IgG1, anti-KIR2DL1 and KIR2DL3, respectively), XA-141 (anti-KIR2DL1 with the same specificity as IgM, EB6), anti-CD4 (HP 2.6). ) And anti-DR (D1.12, IgG2a). Instead of JT3A, HP2.6, and DR1.12, other commercially available mAbs with the same specificity can be used. EB6 and GL183 are commercially available (Beckman Coulter Inc., Fullerton, CA). XA-141 is not commercially available, but mAb EB6 is dissolved as described in Moretta et al. (Moretta et al., J Exp Med 1993; 178: 597-604) as shown in the examples and drawings of the present application. Can be used for contrast reconstruction.

  Cells were stained with the appropriate antibody (30mns at 4 ° C) and then with PE or FITC-conjugated polyclonal anti-mouse antibody (Southern Biotechnology Associates Inc). Samples were analyzed by cytofluorimetric analysis (flow cytometry) on a FACScan instrument (Becton Dickinson, Mountain View, CA).

  In this study, the following NK cell clones were used. CP11, CN5 and CN505 are KIR2DL1-positive clones and are stained with EB6 (IgG1, anti-KIR2DL1) or XA-141 (IgM, anti-KIR2DL1 with the same specificity compared to EB6 antibody). CN12 and CP502 are KIR2DL3-expressing NK clones and stained with GL183 antibody (IgG1, anti-KIR2DL / 3).

The cytolytic activity of NK clones is related to their ability to kill target cells expressing HLA-Cw3 or -Cw4 (HLA positive cell lines known to be sensitive to NK cell lysis). Effector NK cells were assessed by a standard 4 hour ⁵¹ Cr release assay. All targets were used at 5000 cells / well in microtiter 96-well plates. The effector: target ratio is noted in the figure (usually 4 effector / target cells). Cell lysis assays were performed with or without the addition of hybridoma supernatants of the indicated monoclonal antibodies at 1/2 (1: 1) dilution. The procedure was basically the same as described in Moretta et al. (Moretta et al., J Exp Med 1993; 178: 597-604).

Example 2: Preparation of a new mAb
Activated polyclonal or monoclonal human NK cell lines as described by Moretta et al. (Moretta et al., J Exp Med 1990; 172: 1589-1598) with 5 week old Balb / C mice. MAbs were generated by immunization. After different cell fusions, mAbs were first selected for their ability to cross-react with EB6 and GL183 positive NK cell lines and clones. In addition, positive monoclonal antibodies were screened for the ability to reconstitute lysis with EB6 positive or GL183 positive NK clones, respectively, of Cw4 or Cw3 positive targets.

Cells were stained as follows. Cells were stained with a panel of antibodies (1 μg / mL or 50 μL supernatant, 30 mns at 4 ° C.) followed by PE-conjugated goat F (ab ′) ₂ fragment anti-mouse IgG (H + L) or PE-conjugated goat F (ab ′ ₂ ) Fragments Stained with anti-human IgG (Fc gamma) antibody (Beckman Coulter). Cytofluorimetric analysis was performed using Epis XL. Performed on an MCL apparatus (Beckman Coulter).

  One of the monoclonal antibodies, DF200 mAb, was found to react with various members of the KIR family, including KIR2DL1 and KIR2DL2 / 3. Both KIR2DL1-positive and KIR2DL2 / 3-positive NK cells stained brightly with DF200 mAb (FIG. 1).

NK clones expressing one or the other (or both in some cases) of these HLA class I specific inhibitory receptors are used as effector cells for target cells expressing one or more HLA-C alleles did. Cytotoxicity assays were performed as follows. The cytolytic activity of YTS-KIR2DL1 or YTS-Eco (KIR-negative) cell lines was assessed by a standard 4 hour ⁵¹ Cr release assay. Target cells were HLA-Cw4 positive or negative B-EBV cell lines, or 721.221 cells transfected with HLA-Cw4. All targets were used at 3000 cells / well in a microtiter plate. The effector / target ratio is noted in the figure. Cell lysis assays were performed with or without the indicated full length or F (ab ′) ₂ fragments of monoclonal mouse or human antibodies. As expected, KIR2DL1 positive (KIR2DL1 +) NK clones show little cytolytic activity against target cells expressing HLA-Cw4 even in the presence of cytolytic activity, and KIR2DL3 ⁺ NK clones become Cw3 positive targets In contrast, it showed little or no activity. However, in the presence of DF200 mAb (used to mask their KIR2DL receptor), NK clones cannot recognize their HLA-C ligands and express Cw3- or Cw4- Strong cytolytic activity was demonstrated against the target (examples of results are shown in FIGS. 2-6).

For example, the C1R cell line (CW4 ⁺ EBV cell line, ATCC No. CRL-1993) expresses HLA-Cw4 (but not the group 1 HLA-C allotype) and is expressed by the KIR2DL1-positive NK clone (CN5 / CN505) It was not killed. However, this inhibition could be reversed efficiently by using either DF200 or a conventional anti-KIR2DL1 mAb. In ^contrast, KIR2DL2 / 3 + KIR2DL1 ^- NK clones (CN12) expressing phenotype, C1R cells efficiently to kill, this killing was unaffected by the DF200 mAb (Figure 2). Similar results are obtained when KIR2DL2- or KIR2DL3-positive NK clones are used against Cw3-positive targets.

Similarly, the Cw4 + 221 EBV cell line was not killed by KIR2DL1 ⁺ transfected NK cells, but efficiently inhibited by using DF200 mAb, Fab fragment of DF200, or conventional anti-KIR2DL1 mAb EB6 or XA141. I was able to restore it. Also, Cw3 positive 721.221 transfectants were not killed by KIR2DL2 ^{+ +} NK cells, but this inhibition could be reversed by using either DF200 or DF200 Fab fragments. Finally, the Cw3 + 721.221 transfectant was not killed by KIR2DL3 ⁺ NK cells, but this inhibition was originally achieved by using either the DF200 Fab fragment or the conventional anti-KIR2DL3 mAb GL183 or Y249. I was able to bring it back. The results are shown in FIG.

F (ab ′) ₂ fragments were also examined for the ability to reconstitute lysis of Cw4 positive targets. Both the DF200 and EB6 Abs F (ab ′) ₂ fragments can restore inhibition of lysis of Cw4 transfected 221 and Cw4 ⁺ TUBO EBV cell lines by KIR2DL1-transfected NK cells. It was. The results are shown in FIG.

Example 3: Biacore analysis of DF200 mAb / KIR2DL1 and DF200 mAb / KIR2DL3 interactions Production and purification of recombinant proteins KIR2DL1 and KIR2DL3 recombinant proteins were produced in E. coli. PCDM8 clone 47.11 vector (Biassoni et al, Eur J Immunol. 1993; 23: 1083-7) and RSVS (gpt) 183 clone 6 vector (Wagtmann et al, Immunity 1995; 2 respectively) using the following primers: : 439-49 and 1995; 3: 801-809), cDNAs encoding the entire extracellular domain of KIR2DL1 (SEQ ID NO: 23) and KIR2DL3 (SEQ ID NO: 25) were amplified by PCR.

Sense: 5'-GGAATTCCAGGAGGAATTTAAAATGCATGAGGGAGTCCACAG-3 '(SEQ ID NO: 13)
Antisense: 5'-CGGGATCCCAGGTGTCTGGGGTTACC-3 '(SEQ ID NO: 14)
Together with the sequence encoding the biotinylation signal, they were cloned in frame into the pML1 expression vector (Saulquin et al, J Exp Med. 2003; 197: 933-8).

Protein expression was performed in the BL21 (DE3) bacterial strain (Invitrogen). Transfected bacteria were grown in medium supplemented with ampicillin (100 μg / mL) at 37 ° C. until OD ₆₀₀ = 0.6 and expression was induced with 1 mM IPTG.

  The protein was recovered from the inclusion bodies under denaturing conditions (8M urea). Refolding of the recombinant protein was achieved by reducing L-arginine (400 mM, 400 mM, Sigma) and β-mercaptoethanol (1 mM) in 20 mM Tris, pH 7.8, NaCl 150 mM buffer. Reduced and oxidized glutathione (5 mM and 0.5 mM, Sigma, respectively) was added during the 0.5 and 0 M urea dialysis steps. Finally, the protein was dialyzed thoroughly against 10 mM Tris, pH 7.5, NaCl 150 mM buffer. The refolded soluble protein was concentrated and then purified on a Superdex 200 size exclusion column (Pharmacia; AKTA system).

  Surface plasmon resonance measurement was performed with a Biacore apparatus (Biacore). In all Biacore experiments, HBS buffer supplemented with 0.05% surfactant P20 served as the running buffer.

  Protein Immobilization Recombinant KIR2DL1 and KIR2DL3 proteins produced as described above were covalently immobilized to carboxyl groups in the dextran layer on Sensor Chip CM5 (Biacore). The surface of the sensor chip was activated with EDC / NHS (N-ethyl-N ′-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide, Biacore). Protein in coupling buffer (10 mM acetate, pH 4.5) was injected. Inactivation of the remaining activated groups was performed with 100 mM ethanolamine pH 8 (Biacore).

例4： 新規のヒト抗KIR mAbの作製
ヒト抗体レパートリーを発現するように操作されたトランスジェニックマウスを、組換え可溶性ＫＩＲタンパク質で免疫することによって、ヒトモノクローナル抗ＫＩＲ Ａｂを作製した。ハイブリドーマを樹立するための異なる細胞融合の後、まず、固定化されたKIR2DL1及びKIR2DL3タンパク質（上記の例３に記載のとおり産生された）と交叉反応する能力について、mAbsを選択した。１−７Ｆ９、１−４Ｆ１、１−６Ｆ５及び１−６Ｆ１を含む幾つかのヒトモノクローナル抗体は、ＫＩＲ２ＤＬ１及びＫＩＲ２ＤＬ２／３と交差反応されることが見出された。 Affinity measurement. Various concentrations of soluble antibody (1 × 10 ⁻⁷ to 4 × 10 ⁻¹⁰ M) were given on the immobilized samples for kinetic measurements. Measurements were performed at a continuous flow rate of 20 μL / min. For each cycle, the surface of the sensor chip was regenerated with a 5 μL injection of 10 mM NaOH pH11. BIAlogue Kinetics Evaluation program (BIAevaluation 3.1, Biacore) was used for data analysis. Soluble analyte (40 μL at various concentrations) was injected at a flow rate of 20 μL / min onto a dextran layer containing 500 or 540 reflection units (RU) and 1000 or 700 RU of KIR2DL1 and KIR2DL3, respectively, in HBS buffer. . Data are representative of 6 independent experiments. The results are shown in Table 1 below.

Example 4: Generation of a novel human anti-KIR mAb Human monoclonal anti-KIR Abs were generated by immunizing transgenic mice engineered to express a human antibody repertoire with recombinant soluble KIR protein. After different cell fusions to establish hybridomas, mAbs were first selected for their ability to cross-react with immobilized KIR2DL1 and KIR2DL3 proteins (produced as described in Example 3 above). Several human monoclonal antibodies, including 1-7F9, 1-4F1, 1-6F5 and 1-6F1, were found to be cross-reacted with KIR2DL1 and KIR2DL2 / 3.

  The positive monoclonal antibody was further selected for its ability to reconstitute lysis by KIR2DL1-positive NK transfectants against Cw4-positive target cells. NK cells expressing HLA class I specific inhibitory receptors were used as effector cells for target cells expressing one or more HLA-C alleles (FIGS. 5 and 6). Cytotoxicity assays were performed as described above. The effector / target ratio is noted in the figure and the antibody was used at either 10 μg / mL or 30 μg / mL.

As expected, KIR2DL1 ⁺ NK cells showed little cytolytic activity against target cells expressing HLA-Cw4 even when cytolytic activity was present. However, in the presence of 1-7F9 mAb, NK cells are unable to recognize their HLA-C (ie are no longer inhibited by HLA-C molecules) and have strong cytolysis against Cw4 positive targets. Showed activity. For example, the two cell lines tested (HLA-Cw4 transfected 721.221 and CW4 ⁺ EBV cell lines) were not killed by KIR2DL1 ⁺ NK clonal cells, but this inhibition was observed with mAb 1-7F9 or conventional anti-antibodies. By using any one of KIR2DL1 mAb EB6, it was possible to efficiently restore it. Murine mAbs DF200 and Pan2D (NKVSF1) were compared to human mAb 1-7F9. And in all experiments, 1-7F9 was more potent than any other mAbs tested for induction of cytotoxicity by NK cells. In contrast, antibodies 1-4F1, 1-6F5 and 1-6F1 were unable to reconstitute cytolysis by NK cells against Cw4 positive targets.

Example 5: Biacore competitive binding analysis of mouse and human anti-KIR antibodies (Gauthier et al., Journal of Immunology 1999; 162: 41-50; Saunal and van Regenmortel, Journal of Immunology 1995; 183: 33-41) for immobilized KIR2DL1 (900 RU), KIR2DL3 (2000 RU) and KIR2DS1 (1000 RU), mouse anti-KIR2D antibodies DF200, NKVSF1 (Pan2D), gl183 and EB6, and human Epitope mapping analysis was performed with anti-KIR2D antibodies 1-4F1, 1-6F1, 1-6F5 and 1-7F9.

  All experiments were performed at 15 μg / mL with various antibodies injected for 2 minutes in HBS buffer at a flow rate of 5 μL / min. For each pair of antibodies, competitive binding analysis was performed in two steps. In the first step, a first monoclonal antibody (mAb) is injected over the KIR2D target protein, then a second mAb is injected (without removing the first mAb) and the RU value of the second mAb (RU2) was monitored. In the second stage, the second mAb was first injected directly onto the nude KIR2D protein and the mAb RU value (RU1) was monitored. The percent inhibition of binding of the second mAb to the KIR 2D protein by the first mab was calculated by 100 * (1-RU2 / RU1).

  The results are shown in Tables 2, 3 and 4. In Tables 2, 3 and 4, the antibody labeled “first antibody” is listed in the vertical column, and “second antibody” is listed in the horizontal column. For each antibody combination tested, the value of the direct binding level (RU) of the antibody to the chip is listed in the table. In the table, the direct binding of the second antibody to the KIR2D chip is listed at the top of the column, and the value for the binding of the second antibody to the KIR2D chip when the first antibody is present is shown in the column. Listed at the bottom. Listed to the right of each column is the percent inhibition of second antibody binding. Table 2 shows the binding on the KIR2DL1 chip, Table 3 shows the antibody binding to the KIR2DL3 chip, and Table 4 shows the antibody binding to the KIR2DS1 chip.

例6： カニクイザルＮＫ細胞を用いた抗ＫＩＲ ｍＡｂ滴定
カイクイザルから得られたＮＫ細胞への結合能について、抗ＫＩＲ抗体ＮＫＶＳＦ１を検査した。 The competitive binding of mouse antibodies DF200, NKVSF1 and EB6 and human antibodies 1-4F1, 1-7F9 and 1-6F1 to immobilized KIR2DL1, KIR2DL2 / 3 and KIR2DS1 was evaluated. Epitope mapping obtained from experiments using anti-KIR antibody binding to KIR2DL1 (FIG. 7) shows that (a) antibody 1-7F9 competes with EB6 and 1-4F1, but not NKVSF1 and DF200 (B) Next, antibody 1-4F1 competes with EB6, DF200, NKVSF1 and 1-7 F9; (c) NKVSF1 competes with DF200, 1-4F1 and EB6, but with 1-7F9 And (d) showed that DF200 competes with NKVSF1, 1-4F1 and EB6 but not 1-7F9. Epitope mapping obtained from experiments using anti-KIR antibody binding to KIR2DL3 (FIG. 8) shows that (a) 1-4F1 competes with NKVSF1, DF200, gl183 and 1-7F9; (b) 1- 7F9 competes with DF200, gl183 and 1-4F1, but not NKVSF1; (c) NKVSF1 competes with DF200, 1-4F1 and GL183, but not 1-7F9; and (D) DF200 was shown to compete with NKVSF1, 1-4F1 and 1-7F9, but not GL183. Epitope mapping obtained from experiments using anti-KIR antibody binding to KIR2DS1 (FIG. 9) shows that (a) antibody 1-4F1 competes with NKVSF1, DF200 and 1-7F9; (b) 1-7F9 Competes with 1-4F1, but not DF200 and NKVSF1; (c) NKVSF1 competes with DF200 and 1-4F1, but not 1-7F9; and (d) DF200 Showed that it competes with NKVSF1 and 1-4F1, but not 1-7F9.

Example 6: Anti-KIR mAb titration using cynomolgus monkey NK cells The anti-KIR antibody NKVSF1 was examined for its ability to bind to NK cells obtained from cynomolgus monkeys.

  Purification of monkey PBMC and generation of polyclonal NK cell bulk Cynomolgus monkey PBMC were prepared from Sodium citrate CPT tubes (Becton Dickinson). Purification of NK cells was performed by negative depletion (Macaque NK cell enrichment kit, Stem Cell Technology). NK cells are cultured on irradiated human feeder cells with 300 U / mL interleukin 2 (Proleukin, Chiron Corporation) and 1 ng / mL phytohemagglutinin A (Invitrogen, Gibco) to obtain a polyclonal NK cell population It was.

Pan2D mAb Titration with Cynomolgus NK Cells Cynomolgus NK cells (NK bulk 16 days) were incubated with different amounts of Pan2D mAb followed by PE-conjugated Fab ′ ₂ fragment anti-mouse IgG (H + L) antibody Incubated. The percentage of positive cells was determined using an isotype control (purified mouse IgG1). Samples were made in duplicate. Average fluorescence intensity = MFI.

  Antibody binding to monkey NK cells is shown in FIG.

Example 7: Screening for human anti-KIR mAbs Human monoclonal anti-KIR Abs were generated by immunizing transgenic mice engineered to express the human antibody repertoire with recombinant KIR protein (as described in Example 4). Street). To generate cross-reactive anti-KIR antibodies, animals were sequentially immunized with different KIRs expressed in solubilized form as described in Example 3 or on the surface of the cells.

  Next, after fusing different cells, mAbs were first selected for the ability to cross-react with immobilized KIR2DL1 and KIR2DL3 proteins using standard methods in ELISA. Several human monoclonal antibodies, including 1-7F9, 1-4F1, 1-6F5 and 1-6F1, were found to react with all of KIR2DL1, KIR2DL2 and KIR2DL3 by ELISA. MAbs that react with both KIR2DL1 and KIR2DL2 / 3 have been named “KIR2DL-crossreactive mAbs”.

  The KIR2DL cross-reactive mAbs were then examined by flow cytometry for the ability to react with KIR on the cell surface. Briefly, binding of human anti-KIR mAbs, they stably overexpressing KIRs (e.g. YTS-2DL1, BWZ-KIR2DL2 and BWZ-KIR2DS4) or not expressing KIRs ( For example, YTS and BWZ) were examined by incubation with various cell lines. Briefly, cells were incubated with various concentrations of human anti-KIR mAb1-7F9 (typically 0-50 μg / ml) in DMEM medium containing 2% FCS. Following incubation, cells were washed and incubated with an APC-conjugated secondary antibody specific for human IgG. All incubation and washing steps were performed at 0-4 ° C. Subsequently, the cells were washed, resuspended in PBS and analyzed by flow cytometry using a FACScalibur or FACScanto (both from Beckton Dickinson). A typical example is shown in FIG. For example, 1-7F9 and 1-4F1 were found not to bind to cells transfected with KIR2DS4, while NKVSF1 (Pan2D) was found to bind (FIG. 16).

  The ability of human anti-KIR mAbs to block the interaction between KIR and its HLA-C ligand is Tested by reconstitution.

  In biochemical binding experiments, the ability of human anti-KIR mAbs (including 1-7F9) to block the interaction between HLA-C and KIR-molecules, soluble recombinant KIR-Fc fusions The protein was evaluated by competing with binding to cells expressing HLA-C. KIR-Fc protein was produced as described by Wagtmann et al. (Wagmann et al., Immunity 1995; 3 (6): 801-9) (where human Fc was replaced with mouse IgG1 Fc). Soluble KIR-Fc binds to cells expressing a particular HLA-C allotype, which is recognized by KIR2DL1. This binding can then be visualized by flow cytometry using a secondary fluorochrome-bound Ab specific for the mouse Fc portion of the KIR-Fc protein. For example, KIR2DL1-Fc bound to cells transfected with HLA-Cw * 0402 (LCL 721.221-Cw4 transfectant) (Litwin et al., J Exp Med. 1993; 178: 1321-36). However, it did not bind to untransfected LCL721.221 cells (see FIG. 11A). To test whether anti-KIR mAbs block this interaction between KIR2DL1-Fc and HLA-Cw4, preincubate KIR2DL1-FC protein with increasing concentrations of anti-KIR mAbs. Then added to LCL 721.221-Cw4 cells, incubated at 4 ° C., washed, incubated with APC-conjugated anti-mouse IgG1 Fc, washed and flow cytometry using FACScalibur or FACScanto (Beckton Dickinson) And analyzed by standard methods. Some mouse anti-KIR mAbs (eg DF200) and some human anti-KIR mAbs (ie 1-7F9 and 1-4F1) bind to cells in which KIR2DL1-Fc expresses HLA-Cw4 It was shown that these mAbs block the interaction between KIR2DL1 and HLA-Cw4. As an example, FIG. 17A shows that DF200 prevents KIR2DL1-Fc from binding to cells expressing HLA-Cw4. FIG. 17B shows that 1-7F9 mAbs prevent the binding of KIR2DL1 to HLA-Cw4. At the same time, anti-KIR mAbs were tested for their ability to block the binding of KIR2DL2 to HLA-Cw3. Antibodies that block the binding of KIR2DL-Fc to HLA-C-expressing cells in a dose-dependent manner (as illustrated in FIG. 17) are referred to as “blocking mAbs” and are functionally described below. It was further tested in a cytotoxicity assay.

The therapeutic utility of human anti-KIR mAbs is associated with the ability to induce tumor cell killing by KIR-expressing NK cells by blocking KIR-mediated inhibitory signals. Therefore, it was important to evaluate whether human KIR2DL cross-reactivity and blocking mAbs can induce lysis of target cells expressing HLA-C by NK cells expressing KIR2DL. For this purpose, the following experiments were carried out:
^{In a 51} Cr-release cytotoxicity assay, KIR2DL1-expressing YTS cells (YTS-2DL1) can kill LCL 721.221-Cw3, but not LCL721.221-Cw4 cells (FIG. 18A). ). In contrast, YTS effector cells (YTS) deficient in KIRs effectively killed both cell lines. As described above, YTS-2DL1 effector cells were not able to kill LCL 721.221-Cw4 cells (this result is due to HLA-Cw4-induced inhibitory signaling via KIR2DL1). When YTS-2DL1 cells were preincubated with 1-7F9 in a ⁵¹ Cr release cytotoxicity assay, LCL721.221-Cw4 cells were killed in a 1-7F9 concentration-dependent manner (FIG. 18B). 3-1F13 (a human cross-reactive anti-KIR mAb that binds with high affinity to KIR2DL1 but cannot block the interaction between KIR2DL1 and HLA-Cw4) is LCL721. Cannot induce killing of 221-Cw4 cells. Thus, 1-7F9 induces NK-mediated killing of target cells by blocking the KIR-HLA-C interaction between NK- and target cells.

Example 8: Affinity measurement of 1-7F9
The affinity of 1-7F9 for binding to KIR2DL1 and KIR2DL3 was measured by Biacore analysis using soluble recombinant KIR protein produced as described in Example 3. Biacore experiments were performed as described in Example 3 (provided that the antibody used was human mAb 1-7F9).

The results of affinity determination of binding to KIR2DL1 and -3 by 1-7F9 are shown in Table 5.

Example 9: Epitope mapping of 1-7F9 in KIR2DL1 Epitope mapping of a given monoclonal antibody in a given antigen is performed in silico method (if appropriate) with the presence of both the antibody and antigen X-ray structure / It can be obtained by performing protein-protein docking on the homology model.

  Antibody homology models can be obtained by aligning the light and heavy chain sequences, respectively, with a selection of antibodies in which a 3D structure is present. It is possible to calculate the length of six complementarity determining regions (CDRs) and the optimal template is selected from antibodies with the same CDR length. Using standard techniques, a homology model can be constructed from the selected template.

  In the protein-protein docking approach [recently reviewed (Schneidman-Duhovny et al., Curr Med Chem 2004; 11: 91-107)], two surfaces are represented by features on the surface. Features include hydrogen bonding possibilities, charge, and hydrophobicity. In a grid based method, the space is separated into cubes, each cube is given a value according to its position with respect to the surface (inside, surface, outside) and assigned a set of related features It is done. Brute force matching with a scoring function can now be performed by searching for the total degrees of freedom of three translational and three rotational degrees. The translation angle is manipulated by Fast Fourier Transform, and the rotation angle is treated as an individual calculated value within the standard discretization of the rotation space. From top-scored complexes, specific methods such as shape complementarity, van der Waals interactions, hydrophobicity, electrostatics, desolvation, hydrogen bonding, atomic contact energy, Results can be filtered and scored by residue-residue pairing statistics and hydrophilic group pairing. The top-scoring complexes can be evaluated in detail and the interacting residues and thereby the epitope can be identified.

Methods and analysis
Residue and domain nomenclature for KIRs was according to Fan et al. (Nature 1997; 389: 96-100); that is, domain 1 comprises residues 6-101 and domain 2 contained amino acid residues 105-200. Equipped).

  A homology model of 1-7F9 was constructed using 1OM3 as a template. The 1OM3 structure [created by Calarese et al. (Science 2003; 300: 2065, et seq.)] Is a PDB [Protein Data Bank, at World Wide Web (WWW) address rcsb.org/pdb/; 1OM3 VL sequence: sequence No. 34; 1OM3 VH sequence: SEQ ID NO: 35]. Kabat numbering and CDR allocation were used. The overall alignment was good. And since the CDR lengths were the same, the homology model would be accurate enough for use in protein-protein docking experiments.

  The solution of 1NKR.pdb 2000 was obtained by interprotein docking the 1-7F9 antibody homology model to the KIR2DL1 structure. They were filtered for proximity to D183 (from atoms <6 Å CD) and R131 (from atoms <12 Å CZ), and 133 solutions were analyzed in detail.

  The highest and scoring complexes formed were analyzed to identify possible interactions between R131 (KIR) and D100C (heavy chain) and were constrained. A new rotamer for F181 was selected to minimize energy. The model predicted the following: that is the residue that the antibody described on KIR2DL1 (SEQ ID NO: 23): S20 (Hyd, CB) (Hyd Acceptor, OG), E21 (Hyd, CB, CG), M44 (Hyd, SD, CE), F45 (Hyd, CD1, CD1, CZ, CE2), R68 (Hyd donor, NH2), T70 (Hyd, CB, CG2), Q71 (Hyd , CG), L104 (Hyd, CB, CG, CD1, CD2), Y105 (Hyd, CG, CD2, CE2), R131 (Ion, NH2) (Hyd donor, NH2), S133 (Hyd, CA, CB) ( Hyd donor / acceptor, OG), Y134 (Hyd, CD1, CE1), F181 (Hyd, CB, CG, CD1, CD2, CE1, CE2, CZ), and D183 (Hyd, CB) .

Example 10: Biacore competitive binding analysis of mouse and human anti-KIR antibodies
Competitive binding of 1-7F9, Pan2D (NKVSF1), and DF200 was evaluated by the same method as described in Example 5.

  The results are shown in Tables 6 and 7. In Tables 6 and 7, the antibody labeled "first antibody" is listed in the vertical column, and "second antibody" is listed in the horizontal column. . For each antibody combination tested, the percentage inhibition of the second antibody is listed. If the percentage of inhibition for a particular antibody pair exceeds 40%, the antibody was considered competitive regardless of which antibody was used as the first antibody. Table 6 shows the binding to the KIR2DL1 chip. Table 7 shows the binding of antibodies to the KIR2DL3 chip.

例11: HuKIR1-7F9-Fab'との複合体における、KIR2DL1の結晶構造
1-7F9のFab'断片との複合体におけるKIR2DL1の結晶構造は、Ｘ線結晶学で解析され、2.35Åの解像度で示された。結果は、前記抗体が、KIR2DL1と結合した場合に、MHCクラスI分子との結合をブロックすることができることを確認した（図19）。 Briefly, for KIR2DL1 binding, the results show that: DF200 and Pan2D are competitive and 1-7F9 is not competitive with either DF200 or Pan2D . For KIR2DL3 binding, DF200 and Pan2D were competitive, and 1-7F9 was not competitive with Pan2D, but was competitive with DF200.

Example 11: Crystal structure of KIR2DL1 in complex with HuKIR1-7F9-Fab '
The crystal structure of KIR2DL1 in a complex with the Fab ′ fragment of 1-7F9 was analyzed by X-ray crystallography and shown with a resolution of 2.35 mm. The results confirmed that the antibody can block the binding to MHC class I molecules when bound to KIR2DL1 (FIG. 19).

Materials and Methods Extracellular KIR2DL1 (amino acids 1-223 of SEQ ID NO: 23, residue 16 is arginine (R), residue 114 is leucine (L), and additional N-terminal methionine (M) residues And HuKIR1-7F9 Fab ′ (having the light chain sequence of SEQ ID NO: 36 and the heavy chain sequence of residues 1-221 of SEQ ID NO: 37) were mixed with a slight excess amount of KIR2DL1. This complex was then purified on a gel filtration column. The complex was then concentrated to about 13.5 mg / ml. Crystals were grown in 10% PEG6000 and 500 mM citrate buffer (pH 4.2) by the hang drop method. The crystals were flash frozen (flash frozen) in liquid N _2. Crystal data was then collected using a beam line (BL711I, MAX-lab, Lund, Sweden) at 100K with a resolution of 2.35 mm. Data were integrated with the XDS program (Kabsch, J. Appl. Crystallogr. 1993; 26: 795-800). MOLREP program (Bailey, Acta Crystallogr. Sect. D-Biol. Crystallogr. 1994; 50: 760-763) and structure 1RZJ (Fab part 1) and 1NKR (KIR) registered in PDB for structure-determining molecular substitution It was used. Phase improvements were made with the ARP / WARP program (Lamzin and Wilson, Acta Crystallogr. Sect. D-Biol. Crystallogr. 1993; 49: 129-147). Manual corrections to the X-ray derived structural model were made with the QUANTA program (available from Accelrys Inc., San Diego, CA, USA). The refinement was performed with the REFMAC5 computer program of the CCP4 suite. Water molecules were added by the ARP / WARP program. The model includes 143-224 along with residues 6-114 and 124-200 of KIR2DL1, 1-212 of 1-7F9 light chain and 1-136 of 1-7F9 heavy chain. In addition, 330 water molecules were deployed. For the model, R- and R-free were 0.191 and 0.253, respectively.

The resulting contacts were identified by the CCP4 soot CONTACT computer program with a 4.0 Å cutoff distance between the Fab 'and KIR2DL1 molecules. The resulting KIR2DL1 epitope for human 1-7F9 is the residue of KIR2DL1 (SEQ ID NO: 23): L38, R41, M44, F45, N46, D47, T48, L49, R50, I52, F64, D72, Y80 , P87, and Y88 were found (Tables 8 and 9). Residues 1-7F9 involved in the interaction with KIR2DL1 include S28, V29, S30, Y32, S92, N93, and W94 of the 1-7F9 variable light (VL) chain (SEQ ID NO: 15, Table 8) and Variable heavy (VH) chains (SEQ ID NO: 17) T28, F29, S30, F31, I54, F55, E74, S77, G102, S103, Y105, Y106, D107, and Y108 were included. Further, the KIR2DL1 epitope (and the residue involved in hydrogen bonding) is pointed out in the amino acid sequence of KIR2DL1 in FIG. The isotropic replacement factor (also called “temperature factor” or “B value”) obtained from coordinate refinement is the N-terminal domain of KIR2DL1 and the 1-7F9 antibody Fab ′ fragment. Showed relatively low values for the variable domains, all of which were directly involved in intermolecular binding. This showed the following: that the binding force between the two molecules in the crystal complex is strong and stable, and that the crystal structure is stable with the KIR2DL1 / 1-7F9 Fab 'complex. It was shown to support what was shown.

  The 1-7F9 Fab ′ molecule binds to the KIR2DL1 molecule on one side of the C′CFGA ′ β sheet of domain D1, but also touches one E β strand residue side chain of the same domain. Connection to the loop residue of the D1 domain is also important for binding (see Fan et al. (Nature 1997; 389: 96-100) for topology naming conventions). More specifically, the connection consists of the following topological parts of KIR2DL1: β strand C (amino acids L38 and R41), a loop between β strands C and C ′, L2 (M44, F45 and N46), β strand C ′ ( D47, T48, L49, R50 and I52), β-strand E (F64), loop between E and F β-strand, L3 (D72), β-strand F (Y80) and between F and G β-strands (P87 and Y88) This is done for the loop.

  The HLA-Cw4 molecule binds to both the D1 and D2 domains of the KIR2DL1 molecule (Fan et al. Nat. Immunol. 2001; 2: 452-460), whereas the 1-7F9 antibody binds only to the KIR2DL1 D1 domain. However, there is a spatial overlap between bound 1-7F9 and bound HLA-Cw4, which is large enough for the 1-7F9 antibody to successfully replace HLA-Cw4. Using the published structure in complex of KIR2DL1 with HLA-Cw4 (PDB accession code 1IM9), the contact residues between KIR2DL1 and HLA-Cw4 can be calculated with the CONTACT program. In the calculation using a cutoff distance of 4.0 mm, the following is observed: the three KIR2DL1 residues involved in contact with HLA-Cw4 residues are 1-7F9 / KIR2DL1 complex In common with the contact residues in (Tables 8 and 9). These three KIR2DL1 residues are M44, F45 and D72.

例12: 1-7F9の物理的な安定性
ヒト抗体1-7F9の生物物理学的な特性および安定性を試験した。タンパク質の折りたたみおよび二次構造を、円二色性(CD)および動的光散乱(DLS；dynamic light scattering)によるオリゴマー形成および凝集によって試験した。保存条件（2年間、5℃）を模倣するために、タンパク質を37゜Cで14日間撹拌してインキュベーションした。 Without being limited to any particular theory, crystallographic results also point out that 1-7F9 does not bind to the activated NK receptor KIR2DS4 [amino acids N47 in the extracellular portion of the KIR2DS4 sequence (SEQ ID NO: 38) and For V72].

Example 12: Physical stability of 1-7F9 The biophysical properties and stability of human antibody 1-7F9 were tested. Protein folding and secondary structure were examined by oligomerization and aggregation by circular dichroism (CD) and dynamic light scattering (DLS). To mimic the storage conditions (2 years, 5 ° C.), the proteins were incubated with agitation at 37 ° C. for 14 days.

Materials and Methods Sample preparation 2 mg / ml 1-7F9, (a) 50 mM Na-phosphate, 0.001% polysorbate 80 (Sigma, P8074), pH 7.0; (b) 50 mM Na-phosphate, 0.001% polysorbate 80 , pH 7.0, 0.5 mM sucrose; (c) 50 mM citric acid, 0.001% polysorbate 80, pH 3.0; and (d) 50 mM Tris, 0.001% polysorbate 80, pH 8.5.

  Circular dichroism (CD). CD measurements were performed at 25 ° C. at a protein concentration of 2.0 mg / ml on an instrument equipped with a Chirascan circular dichroism spectrometer (Applied Photophysics) equipped with a Peltier element for temperature control. The 1-7F9 sample was present in a cylindrical quartz cell with a 0.1 mm optical path length. Buffer scans were recorded and subtracted for each sample spectrum.

  Dynamic light scattering (DLS). DLS was performed using a Dynapro99 temperature controlled DLS instrument (Protein Solutions Inc.) at a protein concentration of 2.0 mg / ml at 25 ° C. Data analysis was performed using Dynamics software provided with the instrument.

Results On the other hand, molecular size did not change for samples at pH 7.0 after 14 days incubation as assessed by DLS. Both samples were formulated at pH 3.0 and pH 8.5 was highly agglomerated in 14 days.

  CD measurements all characterized the beta structure and revealed the following: the sample formulated at pH 7.0 was their secondary structure through an accelerated study However, the signal for the sample containing only polysorbate 80 as an excipient was slightly reduced. This is probably due to a weak precipitation of the sample (since the overall shape of the spectrum did not change). Samples containing sucrose do not show such a decrease. CD measurements of samples formulated at pH 3.0 and 8.5 show a strong change in over time spectral characteristics, possibly as a result of unfolding or other conformational changes. This will result in a non-functional 1-7F9 protein. The change was observed immediately and was most pronounced at pH 3.0.

  Overall, 1-7F9 retains its physical properties and maintains stability under stress conditions (stirring at 37 ° C), with polysorbate 80 and sucrose as excipients at pH 7.0 To do.

Example 13: Affinity determination of 1-7F9 and 1-4F1 (monovalent binding)
The affinity of 1-7F9 and 1-4F1 in monovalent binding to the KIR2DL1 and KIR2DL3 antigens (as opposed to the bivalent binding affinity in Examples 3 and 8) can be determined by surface plasmon resonance techniques using Biacore3000 .

  Briefly, anti-human IgG (Dako RAHIgG, # 0423) was used with HBS-EP buffer at a flow rate of 20 μL / min. The purified antibody was diluted to 10 μg / mL. For each antibody, KIR2DL1 or KIR2DL3 and buffer were injected. The antigen was tested at a concentration of 2000, 500, 200, 50 or 20 nM. The flow rate was maintained at 20 μL / min. Surface regeneration was achieved with a single 30 s injection of glycine-HCl (pH 1.8).

［発明の例示的な側面］
1. 配列番号15または配列番号39と少なくとも50%同一のアミノ酸配列を含んでいる軽鎖可変領域、および／または配列番号17または配列番号41と少なくとも50%同一のアミノ酸配列を含んでいる重鎖可変領域を具備している抗体。 The results are shown in Table 10. There was a significant shift in baseline for the binding of 1-4F1 to KIR2DL1. Without being limited to any particular theory, this matter represents a buffering effect by the matrix or indeed a binding reaction involving two steps (eg due to conformational changes of the antigen). I will.

Exemplary aspects of the invention
1. a light chain variable region comprising an amino acid sequence at least 50% identical to SEQ ID NO: 15 or SEQ ID NO: 39, and / or a heavy chain comprising an amino acid sequence at least 50% identical to SEQ ID NO: 17 or SEQ ID NO: 41 An antibody comprising a variable region.

  2. a light chain variable region comprising an amino acid sequence at least 80% identical to SEQ ID NO: 15 or SEQ ID NO: 39 and / or a heavy chain comprising an amino acid sequence at least 80% identical to SEQ ID NO: 17 or SEQ ID NO: 41 An antibody comprising a variable region.

  3. a light chain variable region comprising an amino acid sequence at least 90% identical to SEQ ID NO: 15 or SEQ ID NO: 39, and / or a heavy chain comprising an amino acid sequence at least 90% identical to SEQ ID NO: 17 or SEQ ID NO: 41 An antibody comprising a variable region.

  4. A light chain variable region comprising an amino acid sequence at least 95% identical to SEQ ID NO: 15 or SEQ ID NO: 39, and / or a heavy chain comprising an amino acid sequence at least 95% identical to SEQ ID NO: 17 or SEQ ID NO: 41 An antibody comprising a variable region.

  5. A light chain variable region comprising an amino acid sequence at least 98% identical to SEQ ID NO: 15 or SEQ ID NO: 39, and / or a heavy chain comprising an amino acid sequence at least 98% identical to SEQ ID NO: 17 or SEQ ID NO: 41 An antibody comprising a variable region.

  6. The antibody of any preceding aspect, wherein the light chain variable region comprises at least one conserved amino acid substitution, each amino acid substitution at a corresponding position in SEQ ID NO: 15 or SEQ ID NO: 39. Conserved antibodies compared to the amino acids of.

  7. The antibody of any of the preceding aspects, wherein the heavy chain variable region comprises at least one conserved amino acid substitution, each amino acid substitution at a corresponding position in SEQ ID NO: 17 or SEQ ID NO: 41. Conserved antibodies compared to the amino acids of.

8. The antibody according to any one of the preceding aspects, wherein the antibody comprises at least one of the following (a) to (f):
(a) the amino acid sequence of light chain CDR1 at least 90% identical to residues 24-34 of SEQ ID NO: 15;
(b) the amino acid sequence of light chain CDR2 at least 90% identical to residues 50-56 of SEQ ID NO: 15;
(c) the amino acid sequence of the light chain CDR3 that is at least 90% identical to residues 89-97 of SEQ ID NO: 15;
(d) the amino acid sequence of heavy chain CDR1 at least 90% identical to residues 31-35 of SEQ ID NO: 17;
(e) the amino acid sequence of heavy chain CDR2 at least 90% identical to residues 50-65 of SEQ ID NO: 17; and
(f) Amino acid sequence of heavy chain CDR3 that is at least 90% identical to residues 99-112 of SEQ ID NO: 17.

9. The antibody according to any one of the preceding aspects, comprising at least one of the following (a) to (f):
(a) the amino acid sequence of light chain CDR1 corresponding to residues 24-34 of SEQ ID NO: 15;
(b) the amino acid sequence of light chain CDR2 corresponding to residues 50-56 of SEQ ID NO: 15;
(c) the amino acid sequence of light chain CDR3 corresponding to residues 89-97 of SEQ ID NO: 15;
(d) the amino acid sequence of heavy chain CDR1 corresponding to residues 31-35 of SEQ ID NO: 17;
(e) the amino acid sequence of heavy chain CDR2 corresponding to residues 50-65 of SEQ ID NO: 17; and
(f) Amino acid sequence of heavy chain CDR3 corresponding to residues 99-112 of SEQ ID NO: 17.

10. The antibody according to any one of the preceding aspects, wherein the antibody comprises at least one of the following (a) to (f):
(a) the amino acid sequence of light chain CDR1 consisting essentially of residues 24-34 of SEQ ID NO: 15;
(b) the amino acid sequence of light chain CDR2 consisting essentially of residues 50-56 of SEQ ID NO: 15;
(c) the amino acid sequence of light chain CDR3 consisting essentially of residues 89 to 97 of SEQ ID NO: 15;
(d) the amino acid sequence of heavy chain CDR1 consisting essentially of residues 31-35 of SEQ ID NO: 17;
(e) the amino acid sequence of heavy chain CDR2 consisting essentially of residues 50-65 of SEQ ID NO: 17; and
(f) the amino acid sequence of heavy chain CDR3 consisting essentially of residues 99-112 of SEQ ID NO: 17.

  11. The antibody according to any one of aspects 8 to 10, comprising at least two of (a) to (f).

  12. The antibody according to any one of aspects 8 to 10, comprising at least three of (a) to (f).

  13. The antibody according to any one of aspects 8 to 10, comprising at least four of (a) to (f).

  14. The antibody according to any one of aspects 8 to 10, comprising at least 5 of (a) to (f).

  15. The antibody according to any one of aspects 8 to 10, comprising all of (a) to (f).

  16. The antibody according to any one of the preceding aspects, comprising a light chain variable region comprising the amino acid sequence of SEQ ID NO: 15 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 17. antibody.

17. The antibody according to any one of aspects 1 to 7, comprising at least one of the following (a) to (f):
(a) the amino acid sequence of light chain CDR1 at least 90% identical to residues 24-34 of SEQ ID NO: 39;
(b) the amino acid sequence of light chain CDR2 at least 90% identical to residues 50-56 of SEQ ID NO: 39;
(c) the amino acid sequence of light chain CDR3 at least 90% identical to residues 89-97 of SEQ ID NO: 39;
(d) the amino acid sequence of heavy chain CDR1 at least 90% identical to residues 31-35 of SEQ ID NO: 41;
(e) the amino acid sequence of heavy chain CDR2 at least 90% identical to residues 50-66 of SEQ ID NO: 41; and
(f) the amino acid sequence of heavy chain CDR3 at least 90% identical to residues 99-113 of SEQ ID NO: 41.

18. The antibody according to any one of aspects 1 to 7 and 17, comprising at least one of the following (a) to (f):
(a) the amino acid sequence of light chain CDR1 corresponding to residues 24-34 of SEQ ID NO: 39;
(b) the amino acid sequence of light chain CDR2 corresponding to residues 50-56 of SEQ ID NO: 39;
(c) the amino acid sequence of light chain CDR3 corresponding to residues 89-97 of SEQ ID NO: 39;
(d) the amino acid sequence of heavy chain CDR1 corresponding to residues 31-35 of SEQ ID NO: 41;
(e) the amino acid sequence of heavy chain CDR2 corresponding to residues 50-66 of SEQ ID NO: 41; and
(f) Amino acid sequence of heavy chain CDR3 corresponding to residues 99-113 of SEQ ID NO: 41.

19. The antibody according to any one of aspects 1 to 7 and 17 to 18, comprising at least one of the following (a) to (f):
(a) the amino acid sequence of light chain CDR1 consisting essentially of residues 24-34 of SEQ ID NO: 39;
(b) the amino acid sequence of light chain CDR2 consisting essentially of residues 50-56 of SEQ ID NO: 39;
(c) the amino acid sequence of light chain CDR3 consisting essentially of residues 89-97 of SEQ ID NO: 39;
(d) the amino acid sequence of heavy chain CDR1 consisting essentially of residues 31 to 35 of SEQ ID NO: 41;
(e) the amino acid sequence of heavy chain CDR2 consisting essentially of residues 50-66 of SEQ ID NO: 41; and
(f) The amino acid sequence of heavy chain CDR3 consisting essentially of residues 99 to 113 of SEQ ID NO: 41.

  20. The antibody according to any one of aspects 17 to 19, comprising at least two of (a) to (f).

  21. The antibody according to any one of aspects 17 to 19, comprising at least three of (a) to (f).

  22. The antibody according to any one of aspects 17 to 19, comprising at least four of (a) to (f).

  23. The antibody according to any one of aspects 17 to 19, comprising at least five of (a) to (f).

  24. The antibody according to any one of aspects 17 to 19, comprising all of (a) to (f).

  25. The antibody according to any one of aspects 1 to 7 and 17 to 24, wherein the light chain variable region comprises the amino acid sequence of SEQ ID NO: 39, and the heavy chain variable comprises the amino acid sequence of SEQ ID NO: 41 An antibody comprising a region.

  26. The antibody according to any of the preceding aspects, wherein the antibody does not bind to at least one KIR2DS4 and KIR2DS3.

  27. The antibody according to any one of aspects 1 to 15 and 26, which blocks the binding of KIR2DL1 and KIR2DL2 / 3 to HLA-C class I molecules.

  28. The antibody according to any one of aspects 1 to 15 and 26 to 27, which enhances the lytic activity of NK cells against human target cells expressing HLA-C class I molecules.

  29. The antibody according to any one of aspects 1 to 15 and 26 to 28, which enhances the lytic activity of NK cells against human target cells expressing HLA-C class I molecules, more than DF200 Antibodies that are more efficient.

  30. The antibody according to any one of aspects 1 to 15 and 26 to 28, which enhances the lytic activity of NK cells against human target cells expressing HLA-C class I molecules, more than DF200 Antibodies that are more efficient.

  31. The antibody according to any one of aspects 1 to 15 and 26 to 28, which enhances the lytic activity of NK cells against human target cells expressing HLA-C class I molecules, rather than EB6 Antibodies that are more efficient.

  32. The antibody according to any of the preceding aspects, wherein the antibody is a human or humanized antibody.

  33. The antibody according to any of the preceding aspects, wherein the antibody is IgG1, IgG2, IgG3, or IgG4.

  34. The antibody according to any of the preceding aspects, wherein the antibody is human IgG4.

  35. The antibody according to any one of aspects 1 to 34, which is human IgG2.

  36. A human IgG4 antibody comprising a light chain variable region comprising the amino acid sequence of SEQ ID NO: 15 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 17.

  37. A human IgG2 antibody comprising a light chain variable region comprising the amino acid sequence of SEQ ID NO: 39 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 41.

  38. An antibody that competes with 1-7F9 for binding to KIR2DL1 or KIR2DL2 / 3 and is not 1-4F1, DF200, gl183, A210, A803 (g), or EB6.

  39. An antibody that competes with 1-4F1 for binding to KIR2DL1 or KIR2DL2 / 3 and is not 1-7F9, DF200, gl183, A210, A803 (g), or EB6.

  40. The antibody according to aspect 38, wherein the antibody is not 1-4F1.

  41. The antibody according to aspect 39, wherein the antibody is not 1-7F9.

  42. The antibody according to any one of aspects 38 to 39, wherein the antibody is not DF200.

  43. The antibody according to any one of aspects 38 to 39, wherein the antibody is not gl183.

  44. The antibody according to any one of aspects 38 to 39, wherein the antibody is not EB6.

45. The antibody according to any of the preceding aspects, wherein the antibody has a _{Kd of} about 20 nM or less for KIR2DL1.

46. A antibody according to any one of the preceding aspects, antibodies with 10.9nM following a K _d against KIR2DL1.

47. A antibody according to any one of the preceding aspects, antibodies with 0.45nM following a K _d against KIR2DL1.

48. A antibody according to any one of the preceding aspects, an antibody having about 20nM or less a K _d against KIR2DL3.

49. A antibody according to any one of the preceding aspects, antibodies with 2.0nM following a K _d against KIR2DL3.

50. A antibody according to any one of the preceding aspects, antibodies with 0.025nM following a K _d against KIR2DL3.

  51. The human antibody according to any of the preceding aspects.

  52. A human or humanized antibody that competes with 1-7F9 for binding to KIR2DL1.

  53. A human or humanized antibody that competes with 1-4F1 for binding to KIR2DL1.

  54. A human or humanized antibody that competes with 1-7F9 for binding to KIR2DL2.

  55. A human or humanized antibody that competes with 1-4F1 for binding to KIR2DL2.

  56. A human or humanized antibody that competes with 1-7F9 for binding to KIR2DL3.

  57. A human or humanized antibody that competes with 1-4F1 for binding to KIR2DL3.

  58. A human or humanized antibody that competes with 1-7F9 for binding to KIR2DL1 and KIR2DL2 / 3.

  59. A human or humanized antibody that competes with 1-4F1 for binding to KIR2DL1 and KIR2DL2 / 3.

  60. The human or humanized antibody of any of aspects 52-59, wherein the antibody does not bind to at least one KIR2DS4 and KIR2DS3.

  61. A human or humanized antibody according to any of aspects 52-60, wherein the antibody blocks the binding of KIR2DL1 and KIR2DL2 / 3 to HLA-C class I molecules.

  62. The human or humanized antibody according to any one of aspects 52 to 47, which enhances the lytic activity of NK cells against human target cells expressing HLA-C class I molecules.

  63. A human or humanized antibody according to any of aspects 52 to 62, comprising a light chain variable region comprising an amino acid sequence at least 90% identical to SEQ ID NO: 15, and at least 90% identical to SEQ ID NO: 17 A heavy chain variable region comprising at least 90% amino acid sequence, or a light chain variable region comprising at least 90% amino acid sequence identical to SEQ ID NO: 39, and a heavy comprising at least 90% amino acid sequence identical to SEQ ID NO: 41 An antibody comprising a chain variable region.

  64. The human or humanized antibody according to any of aspects 52 to 63, wherein the antibody is IgG1, IgG2, IgG3, or IgG4.

  65. The human or humanized antibody of aspect 64, wherein the antibody is IgG4.

  66. The human antibody according to aspect 64, wherein the antibody is IgG2.

  67. Human or humanized antibodies that bind to each one of KIR2DL1, -2, and -3, and block the binding of KIR2DL1, -2, and -3 to HLA-C class I molecules .

  68. An antibody that interacts with residues M44 and F45 of KIR2DL1.

  69. The antibody of aspect 68, wherein the antibody further binds to one or more KIR2DL1 residues L38, R41, N46, D47, T48, L49, R50, I52, F64, D72, Y80, P87, and Y88.

  70. An antibody that binds to KIR2DL1 only within the region defined by amino acid residues L38, R41, M44, F45, N46, D47, T48, L49, R50, I52, F64, D72, Y80, P87, and Y88.

  71. Binds to KIR2DL1 and KIR2DL2 / 3 and outside the region defined by residues L38, R41, M44, F45, N46, D47, T48, L49, R50, I52, F64, D72, Y80, P87, and Y88 Antibodies that do not interact with any amino acid residues.

  72. The antibody according to any one of aspects 67 to 71, which is a human antibody 1-7F9.

  73. An antibody fragment or antibody derivative comprising at least one light chain CDR of an antibody according to any of the preceding aspects.

  74. An antibody fragment or antibody derivative comprising at least two light chain CDRs of the antibody according to any of the preceding aspects.

  75. An antibody fragment or antibody derivative comprising at least three light chain CDRs of the antibody according to any of the preceding aspects.

  76. An antibody fragment or antibody derivative comprising the VL region of the antibody according to any of the preceding aspects.

  77. An antibody fragment or antibody derivative comprising at least one heavy chain CDR of an antibody according to any of the preceding aspects.

  78. An antibody fragment or antibody derivative comprising at least two heavy chain CDRs of an antibody according to any of the preceding aspects.

  79. An antibody fragment or antibody derivative comprising at least three heavy chain CDRs of an antibody according to any of the preceding aspects.

  80. An antibody fragment or antibody derivative comprising the VH region of the antibody according to any of the preceding aspects.

  81. An antibody fragment or antibody derivative comprising all CDRs of the antibody according to any of the preceding aspects.

  82. An antibody fragment or antibody derivative comprising the VH and VL regions of the antibody according to any of the preceding aspects.

  83. A hybridoma that produces the antibody according to any of the preceding aspects.

  84. A nucleic acid encoding the antibody or antibody fragment according to any one of aspects 1 to 82.

  85. A vector comprising the nucleic acid according to aspect 84.

  86. A cell comprising the vector according to aspect 85.

  87. The cell according to aspect 86, wherein the cell is selected from monkey COS cells, CHO cells, and human myeloma cells.

  88. A method of producing an anti-KIR antibody or antibody fragment comprising culturing the cell of aspect 86 under conditions suitable for the expression of the anti-KIR antibody, antibody fragment, respectively.

  89. A method of producing an anti-KIR antibody or antibody fragment comprising providing an anti-KIR antibody or antibody fragment and attaching at least one derivative component thereto.

  90. A method of treating cancer in a subject comprising administering to a subject suffering from cancer an effective amount of an antibody or antibody fragment or derivative according to any of aspects 1-82. .

  91. The method of aspect 90, further comprising administering to the patient an agent selected from a chemotherapeutic agent, a radiation therapy agent, an anti-angiogenic agent, an immunomodulatory agent, and a hormonal agent.

  92. A method for treating a disease or disorder caused by a virus in a subject, wherein the antibody or antibody fragment or derivative according to any one of aspects 1 to 82 is applied to a subject suffering from a disease or disorder caused by a virus. A method comprising administering an effective amount of.

  93. Use of an antibody or antibody fragment or derivative according to any of aspects 1 to 82 in the preparation of a medicament for treating cancer.

  94. Use of an antibody or antibody fragment or derivative according to any of aspects 1 to 82 in the preparation of a medicament for treating a disease or disorder caused by a virus.

  95. A composition comprising the antibody or antibody fragment or derivative according to any one of aspects 1 to 82 and a pharmaceutically acceptable carrier or excipient.

  96. The composition of aspect 95, comprising polysorbate 80.

  97. The composition of aspect 95, comprising sucrose.

  98. The composition of aspect 95, comprising polysorbate 80 and sucrose.

  99. The composition according to any of aspects 95-98, further comprising an agent selected from a chemotherapeutic agent, a radiation therapy agent, an anti-angiogenic agent, an immunomodulator, and a hormonal agent.

  100. The composition according to any one of aspects 95 to 99, wherein the antibody, antibody fragment, or antibody derivative is covalently bound to a tumor target inducer.

  101. The composition according to any of aspects 95 to 99, wherein the antibody, antibody fragment, or antibody derivative is encapsulated in a liposome.

  102. The composition according to any of aspects 95-99, further comprising an antibody that binds to KIR, wherein the KIR is not KIR2DL1, KIR2DL2, KIR2DL3, KIR2DS1, or KIR2DS2.

  103. A method for increasing the lytic activity of a cell selected from lymphocytes and NK cells, comprising contacting the cells with the antibody, antibody fragment or derivative according to any one of aspects 1 to 82.

  104. A method for reducing the interaction between a KIR expressed by a cell selected from lymphocytes, T cells and NK cells and an HLA-C class I molecule expressed by a target cell, 84. A method comprising contacting a cell with an antibody or antibody fragment or derivative according to any of aspects 1-82.

  105. A method of neutralizing the inhibitory activity of KIR expressed by NK cells, comprising contacting said NK cells with an antibody or antibody fragment or derivative according to any of aspects 1 to 82.

  106. A human antibody that binds to each of KIR2DL1, KIR2DL2, and KIR2DL3, wherein the antibody blocks binding of an HLA-Cw4 molecule to KIR2DL1 and binding of an HLA-Cw3 molecule to at least one KIR2DL2 or KIR2DL3.

  107. The antibody according to any one of aspects 1 to 82, wherein the KIR2DL1 comprises the amino acid sequence of SEQ ID NO: 23, the KIR2DL2 comprises the amino acid sequence of SEQ ID NO: 24, and / or the KIR2DL3 comprises the amino acid sequence of SEQ ID NO: 25 Or an antibody fragment or derivative.

  108. A compound that binds to substantially the same KIR2DL1 epitope as the antibody, antibody fragment, or antibody derivative of any of aspects 1-82.

  109. A compound that binds to essentially the same KIR2DL1 epitope as the antibody, antibody fragment, or antibody derivative of any one of aspects 1-82.

  110. An antibody or antibody derivative comprising a light chain comprising the sequence of SEQ ID NO: 36.

  111. An antibody or antibody derivative comprising a heavy chain comprising the sequence of SEQ ID NO: 37.

  112. An antibody or antibody derivative comprising a light chain on side 109 and a heavy chain on side 110.

  113. An antibody comprising a light chain comprising the sequence of SEQ ID NO: 36 and a heavy chain comprising the sequence of SEQ ID NO: 37.

* * *
All references cited in this specification, including publications, patent applications and patents, are hereby incorporated by reference as if each reference was individually and specifically incorporated by reference. To the same extent, it is incorporated herein by reference.

  All headings and subheadings used herein are for convenience only and should not be construed as limiting the invention in any way.

  Any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted.

  The terms “a” and “an” and “the” and similar referents used in describing the present invention are not specifically described herein, or are clearly opposite. If not, it should be construed to include both singular and plural.

  The citation of a range of values herein is intended only to serve as a shorthand for individually representing each individual value within the range, and Unless otherwise stated, each individual value is incorporated herein as if it were individually described herein. Unless otherwise noted, all exact values described herein are representative of the corresponding approximate values (eg, described with respect to specific factors or measurements). All exact typical values being given can also be considered to represent the corresponding approximate measurements, optionally modified as “about”).

  Unless otherwise stated herein or explicitly stated to the contrary, all methods described herein may be performed in any suitable order.

  Any and all examples or exemplary terms (such as “for example”) described herein are intended merely to better describe the invention and, unless otherwise indicated, Does not limit the scope of Unless expressly stated otherwise, the language herein should not be construed as implying that any element is essential to the practice of the invention.

  The citation and incorporation of patent documents herein is done for convenience purposes only, and the validity, patentability, and / or enforcement of such patent documents. It does not reflect any view of enforceability.

  Unless stated otherwise specifically stated to the contrary, "comprising", "having", "including" or "containing" with respect to an element It is to be understood that any term or embodiment of the invention is described herein using terms such as “consists of”, “consists essentially of” Intended to provide support for similar aspects or embodiments of the invention (eg, a composition described herein as comprising specific elements). Is to be understood as describing a composition of its components, unless otherwise stated herein or clearly stated to the contrary).

  This invention includes all modifications and equivalents of the subject matter recited in the aspects or claims presented herein to the maximum extent permitted by applicable law.

FIG. 1 illustrates the murine monoclonal antibody DF200 that binds to common determinants of various human KIR2DL receptors. (A) Clone CP11, KIR2DL1 +, DF200. (B) Clone CP502, KIR2DL3 +. (C) Clone CP11, KIR2DL1 +, anti-KIR2DL1. (D) Clone CP502, KIR2DL3 +, anti-KIR2DL2 / 3. FIG. 2 illustrates the murine monoclonal antibody DF200 neutralizing KIR2DL1-mediated inhibition of NK cell cytotoxicity against Cw4-expressing target cells, whereby C1R-Cw4 target with anti-KIR mAb Reconstitution of lysis with an effector / target (E / T) ratio of 4/1 is shown. FIG. 3 shows KIR2DL1-mediated inhibition of cytotoxicity against mouse monoclonal antibody DF200, Fab fragment of DF200, and Cw4-positive target cells, and KIR2DL2 / 3-mediated cytotoxicity of NK cells against Cw3-positive target cells. Shown are conventional mouse antibodies specific for either KIR2DL1 (mAbs EB6 and XA-141) or KIR2DL2 / 3 (mAbs GL183 and Y249) neutralizing inhibition. (A) and (C) KIR2DL1 +, target cell 721.221-Cw4 +. (B) KIR2DL2 +, target cell 721.221-Cw3 +. (D) KIR2DL3 +, target cell 721.221-Cw3 +. FIG. 4 illustrates the reconstitution of cell lysis by NK cells expressing KIR2DL1 against HLA Cw4 positive target cells in the presence of F (ab ′) ₂ fragments of DF200 and EB6 antibodies. (A) Target cell 721.221-Cw4; E / T ratio = 1. (B) Target cell B-EBV cell expressing Cw4, E / T ratio = 2. FIG. 5 shows cross-reactive mouse (DF200 and NKVSF1 (Pan2D)) and human (1-7F9, 1-4F1, 1-6F5 and 1-6F1) mAbs, as well as mouse conventional KIR2DL1-specific mAb (EB6 ) Induced NK-mediated killing. Said mAbs induce (reconstitute) killing of 721.221 target cells transfected with HLA-Cw4 by KIR2DL1-expressing NK cells (YTS-KIR2DL1). E / T ratio = 1. (A) 30 μg / ml mAb. (B) 10 μg / ml mAb. FIG. 6 illustrates the induction of NK-mediated killing by the same antibody as in FIG. The mAbs induce (reconstruct) killing of B-EBV cells expressing endogenous HLA-Cw4 by KIR2DL1-expressing NK cells (YTS-KIR2DL1). E / T ratio = 2. (A) 30 μg / ml mAb. (B) 10 μg / ml mAb. FIG. 7 illustrates an epitope map showing the results of competitive binding experiments obtained by surface plasmon resonance (BIAcore®) analysis using an anti-KIR antibody against KIR2DL1, with the overlapping circles representing KIR2DL1 Represents the overlap between antibodies in binding to. The results show that 1-7F9 competes with EB6 and 1-4F1 for KIR2DL1, but does not compete with NKVSF1 (Pan2D) and DF200. Antibody 1-4F1 then competes with EB6, DF200, NKVSF1 (Pan2D), and 1-7F9. Antibody NKVSF1 (Pan2D) competes with DF200, 1-4F1, and EB6 for KIR2DL1, but not 1-7F9. DF200 competes with NKVSF1 (Pan2D), 1-4F1, and EB6 for KIR2DL1, but not 1-7F9. FIG. 8 illustrates an epitope map showing the results of a competitive binding experiment obtained by BIAcore® analysis using an anti-KIR antibody against KIR2DL3, with the overlapping circles representing over-binding to KIR2DL3. Represents a lap. The results show that 1-4F1 competes with NKVSF1 (Pan2D), DF200 and gl183, and 1-7F9 for KIR2DL3. 1-7F9 competes with DF200, gl183, and 1-4F1 for KIR2DL3, but not NKVSF1 (Pan2D). NKVSF1 (Pan2D) competes with DF200, 1-4F1, and GL183 for KIR2DL3, but not 1-7F9. DF200 competes with NKVSF1 (Pan2D), 1-4F1, and 1-7F9 for KIR2DL3, but not with GL183. FIG. 9 illustrates an epitope map showing the results of a competitive binding experiment obtained by BIAcore® analysis using an anti-KIR antibody against KIR2DS1, with the overlapping circles representing an over-binding in KIR2DS1. Represents a lap. The results show that antibody 1-4F1 competes with NKVSF1 (Pan2D), DF200, and 1-7F9 against KIR2DS1. Antibody 1-7F9 competes with 1-4F1 for KIR2DS1, but not with DF200 and NKVSF1 (Pan2D). NKVSF1 competes with DF200 and 1-4F1 for KIR2DS1, but does not compete with 1-7F9. The DF 200 competes with NKVSF1 and 1-4F1 for KIR2DS1, but does not compete with 1-7F9. FIG. 10 illustrates Pan2D (NKVSF1) mAb titration demonstrating mAb binding to cynomolgus monkey NK cells. Cynomolgus monkey NK cells (NK bulk day 16) were incubated with different amounts of NKVSF1 (Pan2D) mAb followed by PE-conjugated Fab ′ ₂ fragment anti-mouse IgG (H + L) antibody Incubated with. The percentage of positive cells was determined using an isotype control (purified mouse IgG1). Samples were made in duplicate. Average fluorescence intensity = MFI. FIG. 11 shows binding of soluble KIR2DL1 and KIR2DL1 (R131W) mutants to cells. (A) Binding of increasing concentrations of soluble KIR2DL1-Fc and KIR2DL1 (R131W) -hFc mutants to cells expressing HLA-Cw3 or -Cw4. Bound KIR-Fc protein was detected using a secondary fluorochrome-conjugated antibody and revealed by flow cytometry. Average fluorescence is shown on the y-axis. (B) Binding of the indicated anti-KIR mAbs [GL183, EB6, DF200, and Pan2D (NKVSF1)] to KIR2DL1-Fc and 2DL1 (R131W) mutant proteins (human Ig was used as a control). The figure shows that DF200 and Pan2D (NKVSF1) bind to the mutant to a lesser extent than wild type KIR2DL1-Fc. This indicates that both mAbs are affected by the R131W mutation. Therefore, R131 is one of the residues constituting the epitope for DF200 and Pan2D in KIR2DL1. FIG. 12 provides a comparative alignment of the amino acid sequences of the light chain variable region and light chain CDRs of antibodies DF200 and Pan2D (NKVSF1). (A) Alignment of anti-KIR variable light (VL) regions of DF200 (SEQ ID NO: 1) and Pan-2D (SEQ ID NO: 2). The amino acid sequence number above specifies the position for initiation of Met (+1) translation in immature (non-secretory) immunoglobulins. (B) CDR-L1 sequence alignment. Previous residue: usually Cys. After residue: Trp. Typically, Trp-Tyr-Leu. Chain length: 10-17aa. (C) CDR-L2 sequence alignment. Previous residue: generally Ile-Tyr. Chain length: 7aa. Start: Approximately 16aa after the end of CDR-L1. Start: about 24aa from the beginning of the secreted protein. (D) Alignment of CDR-L3 sequences. Previous residue: Cys. Last residue: Phe-Gly-XXX-Gly. Chain length: 7-11aa. Start: about 33aa after the end of CDR-L2. FIG. 13 represents the heavy chain variable region and heavy chain CDRs of antibody DF200. (A) DF-200 VH region, immature protein. The secreted mature form of VH begins at position 20: residue Q. The VH region ends with residue S followed by a constant region (not shown). (B) CDR-H1. Previous residue: Cys-XXX-XXX-XXX. After residue: Trp. Generally, Trp-Val or Trp-Ile. Chain length: 10-14aa. Start: about 22-26aa from the beginning of the secreted protein. (C) CDR-H2. Previous residue: Leu-Glu-Trp-Ile-Gly, but other variations are possible. Subsequent residues: Lys or Arg / Leu or Ile or Val or Phe or Thr or Ala / Thr or Ser or Ile or Ala. Chain length: 16-20aa. Start: Approximately 15aa after the end of CDR-H1. (D) CDR-H3. Previous residue: Cys-XXX-XXX (typically Cys-Ala-Arg). Later residue: Trp-Gly-XXX-Gly. Chain length: 3-25aa. Start: Approximately 33 after the end of CDR-H2. FIG. 14 illustrates the nucleotide and amino acid sequences of the VH and VL sequences of human antibody 1-7F9. (A) Translation of HuKIR 1-7F9 mature variable light chain. (B) Nucleotide sequence encoding HuKIR 1-7F9 mature variable light chain. (C) Translation of HuKIR 1-7F9 mature variable heavy chain. (D) Nucleotide sequence encoding HuKIR 1-7F9 mature heavy chain. FIG. 15 shows VH and VL of monoclonal antibodies 1-7F9, DF200 (VH sequence: SEQ ID NO: 19; VL sequence: SEQ ID NO: 21), and Pan2D (NKVSF1; VH sequence: SEQ ID NO: 20; VL sequence: SEQ ID NO: 22). The amino acid sequence of the sequence is shown. CDRs are shown in boxes. FIG. 16 shows flow cytometry data. This figure shows the binding of mouse antibody Pan2D (NKVSF1) and human mAbs 1-7F9 and 1-4F1 to cells transfected with KIR2DL1, KIR2DL3, KIR2DS1, KIR2DS2, KIR2DS3 or KIR2DS4, as indicated ( Purified antibody: 1 μg / ml). (A)-(R) NKVSF1 (pan2D), 1-7F9, and 1-4F1 all bind to KIR2DL1, KIR2DL3, KIR2DS1, and KIR2DS2. None of the antibodies bound to KIR2DS3. NKVSF1 (not 1-7F9 or 1-4F1) binds to KIR2DS4. FIG. 17 illustrates the results of FACS analysis. (A) Binding of soluble KIR-Fc to HLA-Cw4 on cells is neutralized via antibodies [detected by flow cytometry (FACS)]. FACS-measurement of KIR2DL1-mFc binding to LCL721.221-Cw4 cells was performed after pre-incubation with various concentrations of cross-reactive mAb DF200 or KIR2DL2 / 3-specific mAb GL183. DF200 (not GL183) reduces the binding of KIR2DL1 to HLA-Cw4. Measurements were plotted as a percentage of KIR2DL1-hFc-binding in the absence of inhibitory antibody. (B) FACS-measurement of KIR2DL1-mFc binding to LCL721.221-Cw4 cells was performed after pre-incubation with various concentrations of 1-1F4 and 1-7F9. Measurements were plotted as a percentage of KIR2DL1-mFc-binding in the absence of inhibitory antibody. FIG. 18 illustrates the results of a ⁵¹ Cr-release cytotoxicity assay. (A) LCL721.221-Cw3 cells are efficiently killed at various E: T ratios by both YTS cells (diamonds) and YTS-2DL1 cells (triangles). In contrast, LCL721.221-Cw4 cells are efficiently killed by YTS cells (squares), but are not killed by YTS-2DL1 cells (crosses) due to KIR restriction. (B) In the absence of antibody, YTS-2DL1 cells cannot kill LCL-721.221-Cw4 (E: T ratio 12: 1). 1-7F9 induces killing of LCL721.221-Cw4 cells by YTS-2DL1 cells in a dose-dependent manner. FIG. 19 illustrates the superposition of two crystal complex structures; KIR2DL1 / 1-7F9 Fab ′ and KIR2DL1 / MHC class I, PDB-code 1IM9 (Fan et al., Nat. Immunol. 2001; 2: 452 -460) is illustrated using the common KIR molecule Cα atom (labeled “KIR”) as a template. KIR2DL1 / 1-7F9 Fab 'is shown in a gray linear ribbon style, while KIR2DL1 / MHC class I is shown in a dark tube style. 1-7F9 Fab ′ is labeled “1-7F9” and MHC class I is labeled “MHC”. An overlap between MHC and Fab ′ was observed in the overlay, demonstrating the ability of 1-7F9 Fab ′ to block MHC class I binding to the KIR2DL1 receptor. See Example 11. FIG. 20 shows the binding epitope of 1-7F9 on KIR2DL1 as indicated in the KIR2DL1 sequence. Amino acids within a distance of 4.0 mm from 1-7F9 were highlighted in gray on a black background. Amino acids highlighted with a black background are involved in hydrogen bonding to 1-7F9.

Claims (29)

Killer lg-like receptors (KIR) 2DL1, bind to their KIR2DL2 and KIR2DL3, the KIR2DS4 not bind, reduce the inhibition of mediated NK cytotoxicity by KIR2DL1, KIR2DL2 and KIR2DL3, neutralized or reversed, the KIR2DS4 An isolated humanized or humanized antibody that does not reduce, neutralize or reverse the inhibition of mediated NK cytotoxicity . 2. The isolated human or humanized antibody of claim 1, further not binding to KIR2DS3. 3. The isolated human antibody or humanized antibody of claim 1 or 2, wherein the isolated human antibody blocks binding of at least one KIR2DL1, KIR2DL2 and KIR2DL3 to an HLA-C class I molecule or Humanized antibody . 4. An isolated human or humanized antibody according to any one of claims 1-3, wherein the HLA-Cw4 molecule binds to KIR2DL1, and the HLA-Cw3 molecule binds to at least one KIR2DL2 or KIR2DL3. An isolated humanized or humanized antibody that blocks the binding of. The isolated human antibody or humanized antibody according to any one of claims 1 to 4, which enhances the lytic activity of NK cells against human target cells expressing HLA-C class I molecules. Isolated human antibody. The isolated human antibody or humanized antibody according to any one of claims 1 to 5, wherein the amino acid sequence represented by SEQ ID NO: 15 is bound to at least one of KIR2DL1, KIR2DL2, and KIR2DL3. An isolated human antibody or human that competes with an antibody comprising a light chain variable region comprising a polypeptide having and a heavy chain variable region comprising a polypeptide having the amino acid sequence represented by SEQ ID NO: 17 Antibody . An isolated human or humanized antibody according to any one of claims 1-6, in binding to each KIR2DL1, KIR2DL2 and KIR2DL3, having the amino acid sequence represented by SEQ ID NO: 15 isolated human or humanized antibody that competes with an antibody comprising a heavy chain variable region comprises a polypeptide having the amino acid sequence represented by light chain variable region SEQ ID NO: 17 containing the polypeptide . The isolated human antibody or humanized antibody according to any one of claims 1 to 7, comprising a light chain variable region comprising a polypeptide having the amino acid sequence represented by SEQ ID NO: 15. An isolated human or humanized antibody . The isolated human antibody or humanized antibody according to any one of claims 1 to 8, comprising the following (a) to (c):
(a) a heavy chain CDR1 region consisting of a polypeptide having an amino acid sequence corresponding to residues 31 to 35 of SEQ ID NO: 17;
(b) a heavy chain CDR2 region consisting of a polypeptide having an amino acid sequence corresponding to residues 50-65 of SEQ ID NO: 17; and
(c) a heavy chain CDR3 region consisting of a polypeptide having an amino acid sequence corresponding to residues 99 to 112 of SEQ ID NO: 17. The isolated human antibody or humanized antibody according to any one of claims 1 to 9, comprising a heavy chain variable region comprising a polypeptide having the amino acid sequence represented by SEQ ID NO: 17. An isolated human or humanized antibody . A human or humanized antibody isolated according to any one of claims 1 to 10, a human antibody or human isolated with 0.45nM following dissociation constant (Kd) with respect to KIR2DL1 Antibody . An isolated human or humanized antibody according to any one of claims 1 to 11, isolated human or humanized antibody having the following Kd 0.025 nM against KIR2DL3. The isolated human antibody or humanized antibody according to any one of claims 1 to 12, wherein the amino acid residues L38, R41, M44, F45, N46, D47, T48, L49, R50, I52, An isolated humanized or humanized antibody that binds to a KIR2DL1 epitope comprising F64, D72, Y80, P87, and Y88. The isolated human antibody or humanized antibody according to any one of claims 1 to 13 , wherein the residue M44 of the extracellular portion of the KIR2DL1 polypeptide having the amino acid sequence represented by SEQ ID NO: 23 , An isolated humanized or humanized antibody that blocks the binding of HLA-Cw4 molecules to F45 and D72. 15. An isolated human antibody or humanized antibody according to any one of claims 1-14, wherein the antibody is a monoclonal antibody . 16. An isolated human antibody or humanized antibody according to any one of claims 1-15, wherein the antibody is an IgG1, IgG2, IgG3, or IgG4 antibody . Antibodies . The isolated human antibody or humanized antibody according to any one of claims 1 to 16, wherein the antibody is a human IgG4 antibody . 18. An isolated nucleic acid encoding the human antibody or humanized antibody of any one of claims 1-17. A vector comprising the nucleic acid according to claim 18. A cell comprising the vector according to claim 19. 21. A method of producing a human anti- or humanized anti- KIR antibody comprising culturing the cell of claim 20 under conditions suitable for expression of the anti-KIR antibody. Of any one human or humanized produced by the method described in a human antibody or a humanized antibody or claim 21 in Section antibody of claim 1 to 17, capable of detecting the cytotoxicity of NK cells in a patient A pharmaceutical composition comprising an amount effective to enhance to a certain extent and a pharmaceutically acceptable carrier or excipient. 23. The pharmaceutical composition of claim 22, comprising polysorbate 80, sucrose, or a combination thereof. 24. A pharmaceutical composition according to claim 22 or 23 for use to enhance NK cell activity in a subject. 25. The pharmaceutical composition of claim 24, wherein the subject is a patient suffering from cancer. 26. The pharmaceutical composition of claim 25, wherein the patient suffers from a cancer selected from acute myeloid leukemia, chronic myelogenous leukemia, multiple myeloma, and non-Hodgkin lymphoma. 26. The pharmaceutical composition of claim 25, wherein the patient suffers from a cancer selected from colorectal cancer, kidney cancer, ovarian cancer, lung cancer, breast cancer, and malignant melanoma. object. 25. The pharmaceutical composition of claim 24, wherein the subject is a patient suffering from an infection. 29. The pharmaceutical composition according to any one of claims 24-28, wherein the second antibody binds to an immunomodulator, a hormonal agent, a chemotherapeutic agent, an anti-angiogenic agent, an apoptotic agent, or an inhibitory KIR. A pharmaceutical composition further comprising administering a therapeutic agent selected from: an anti-infective agent, a target inducer, and an adjunct compound.

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